Methods of Mapping Polymorphisms and Polymorphism Microarrays

ABSTRACT

Described herein are methods for the high-throughput discovery and genotyping of nucleotide polymorphisms in DNA, including single nucleotide polymorphism (SNPs) and short deletions and insertions. These methods take advantage of the fact that differences in DNA sequence result in the differential presence of restriction endonuclease digestion sites. Differences can be detected between individuals, or the relative presence detected in a population. Provided approaches involve isolation of short DNA fragments (“tags) near restriction endonuclease sites. The presence of one (or two) of these tags indicates that a site was present. Distinguishable labeling of tags from two individual or populations allows comparative presence of these sites to be assayed on a platform that employs a collection of nucleic acids. Other approaches depend on the differential presence of restriction endonuclease sites, but involve mixing genomic DNA from the two individuals. Regions of DNA with a restriction site in only one individual create an opportunity for primer extension to produce labeled material, which can be assayed on a platform that employs a collection of nucleic acids. Any of a variety of detection platforms can be use with the described approaches. By way of example, highly efficient variant detection microarrays and bead libraries are provided that contain genomic tags with different representations between two populations, so that most elements in the collection of nucleic acids contain an informative SNP between the populations of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/679,693, filed May 10, 2005, and U.S. Provisional Application No.60/782,424, filed Mar. 14, 2006, both of which are incorporated hereinin their entirety.

FIELD

This disclosure relates to methods, materials, and devices fordetecting, resolving, and mapping polymorphisms and genetic differences.It relates to detection of large numbers of polymorphisms, includingspecifically single nucleotide and other short polymorphisms.Representative methods employ differential enzyme digestion andhybridization.

BACKGROUND OF THE DISCLOSURE

Genetic variation exists between different individuals of a species. Forsome organisms, a single nucleotide polymorphism (SNP) may occur every100 basepairs, while other species may have rates greater than onechange in 1000 (Sachidanandam et al., Nature 409(6822): 928-33, 2001).Small (short) nucleotide insertions and deletions may occur at similarfrequencies. While such polymorphisms can complicate some forms ofgenetic analysis, they can also be harnessed to map the inheritance ofchromosomal regions. In model organisms, SNPs have been used to map thelocation of mutations from genetic screens in recombinant progeny(Berger et al., Nat Genet. 29(4): 475-81, 2001; Martin et al., GenomeBiol 2(9): RESEARCH 0036, E-pub Aug. 30, 2001; Wicks et al., Nat Genet.28(2): 160-4, 2001; Stickney et al., Genome Res 12(12): 1929-34, 2002),and to identify the location of phenotypic modifiers in quantitativetrait locus mapping (QTL). In humans, SNPs have been used to identifydisease alleles and phenotypic modifiers in association studies (Bader,Pharmacogenomics 2(1): 11-24. 2001; Pharoah et al., Nat Rev Cancer4(11): 850-60, 2004).

The power of using SNPs increases with the number of SNPs identified,and methods for genotyping individuals for the presence of particularSNPs have improved. In sequenced organisms, bioinformatic approaches ofcomparing expressed sequence tag (EST) data have yielded a wealth ofpotential SNPs (Marth et al., Nat Genet. 23(4): 452-6, 1999; Buetow etal., Proc Natl Acad Sci USA 98(2): 581-4, 2001; Hu et al.,Pharmacogenomics J 2(4): 236-42, 2002). More recently, high-throughputapproaches using high-density oligonucleotide arrays have been employedfor SNP discovery (Matsuzaki et al., Genome Res 14(3): 414-25, 2004).However, these approaches can only be used to study organisms with awell-developed genomics infrastructure and prior knowledge of genome orEST sequence, at significant cost.

Likewise, high-resolution SNP maps have been generated by comparativegenome sequencing of lab populations of interest, such as the commongenetic screen lines FRT 82 and rucuca in Drosophila (Berger et al., NatGenet 29(4): 475-81, 2001; Martin et al., Genome Biol 2(9): RESEARCH0036, E-pub Aug. 30, 2001). These SNP maps are optimized for the linestested, although some proportion of SNPs from the tested populations areexpected to be present in other fly lines as well. The effort involvedin creating these maps makes it unlikely that many additional lines ofinterest will have SNPs discovered at high density in the near future bycomparative sequencing, despite the need for many lines of differentgenetic backgrounds for optimal isolation and recovery of mutations ofinterest.

A frequent objective of previous SNP discovery screens was to identifySNPs that disrupted restriction endonuclease recognition sites.Disruption of such a site allowed for low-cost and rapid genotyping ofthe potential SNP from different individuals, as the read-out was thedifferential digestion of the SNP region. More recently, the capture andsequencing of genomic regions around restriction sites has been used tosample genomes and determine areas of DNA duplication in cancer andmicrobial population dynamics (Wang et al., Proc Natl Acad Sci USA99(25): 16156-61, 2002; Zabarovslca et al., Nucleic Acids Res 31(2):E5-5, 2003). In these approaches, SNPs have been confounding factorsrather than the objective of the techniques, in that SNPs causeuncertainty in the assignment of the short sequence reads to theirproper position in the genome. Other techniques have been used todistinguish the relatedness of individual organisms within a species(see, e.g., U.S. Pat. No. 5,713,258).

While the ability to detect nucleotide polymorphisms has improvedrapidly, it is not routine to detect large number of polymorphismsbetween two individuals, particularly in organisms lacking thoroughgenomic and cDNA sequence information.

SUMMARY OF THE DISCLOSURE

Provided herein in various embodiments are new methods for the routinedetection of polymorphisms (including SNPs and short deletions orinsertions, and other variants), and the creation of new types ofnucleic acid element collections (including microarrays and beadlibraries) that optimize the detection of polymorphisms from thesemethods.

Methods described herein demonstrate the use of restriction site tagsfor single nucleotide polymorphism (SNP) discovery and mutation andvariant mapping.

In one example method, fragments (tags) near restriction sites areisolated from genomic DNA. In a working embodiment, the fragments (tags)are about 1 kb in length. In individuals where nucleotide polymorphismsdisrupt the restriction site, the associated tag will be absent from therestriction site tags selected (in that it will not be the expectedsize). Hybridization of labeled restriction site tags (also referred toas restriction site associated DNA, or RAD) from two differentindividuals to a collection of nucleic acid elements (for instance, in amicroarray or a bead library) allows for polymorphism discovery at arrayelements with differential hybridization between the two individuals.Strategies for optimizing this protocol and using the resulting SNPinformation in mapping mutations are also described.

Yet further provided methods demonstrate the use of extension frommismatched restriction sites for polymorphism discovery and mapping.

In an example of such methods, restriction enzyme-digested genomic DNAfrom two individuals are mixed and annealed. If nucleotide polymorphismsdisrupt a restriction site in one of the two individuals, then the siteof polymorphism will create two short fragments (from one individual)bound to a longer, uncut fragment (from the other individual). One ofthe short fragments can be used as a primer for extension by DNApolymerase, allowing the incorporation of label (e.g., fluorescentdye-linked nucleotides) near the polymorphism site. Hybridization of theresultant labeled DNA to a collection of nucleic acids (e.g., in amicroarray or bead library) allows for SNP discovery at nucleic acidelements with strong hybridization signal intensity. Strategies foroptimizing this protocol and using the resulting SNP information inmapping mutations are also described.

Also described are restriction site tag collections, including forinstance microarrays and bead libraries. These collections, arrays andlibraries have particular utility for detecting polymorphism discovered,for instance, by the methods described herein.

It is acknowledged and recognized that the polymorphism detection anddiscovery methods described herein can be used with variety of platformsfor the detection of polymorphisms (e.g., SNPs and small deletions orinsertions) and generally for nucleic acid variations between twosamples. For projects involving a large amount of SNP discovery ormapping, there are benefits to using a restriction site genomic tagarray or bead library as described herein. A genomic tag array or beadlibrary contains elements that consist of DNA flanking sites ofdigestion for a particular restriction enzyme in the genome. Thus, whenthe experimental and array restriction sites match, each element of thecollection is capable of detecting a restriction site tag (in someembodiment methods), or an extension from restriction site polymorphism(in other embodiment methods). By way of example, such restriction sitegenomic tag arrays provided higher rates of SNP discovery and higherresolution mapping. Also described are subtracted restriction sitegenomic tag collections (e.g., arrays and bead libraries), wherein thearray or library contains (substantially) only elements that differbetween two individuals (or two reference samples). This is possiblewhen the tags from the two individuals (or reference samples) undergo around of subtractive hybridization, a procedure that removes DNA incommon between the two samples. In such subtractive genomic tag arraysand libraries, each element could assay a polymorphism betweenindividuals (or an individual and one or both reference samples).

An advantage of the techniques described herein is that it is possibleto discover a large number of SNPs in the exact population of interest,using only the genomic DNA and a few restriction endonucleases. Thus,researchers need no longer be confined to working with genotypes whereSNP maps have already been developed, or even to organisms withsequenced genomes.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of a representative method for SNP detection byhybridization of restriction site tags to a microarray.

FIG. 2 illustrates identification of recombination breakpoints.Hybridization of tags from FRT 82 and rucuca identify potential SNPsalong the chromosome (top, vertical lines). Hybridization of therecombinant versus FRT 82 will produce tag differences wherever therecombinant chromosome contains rucuca material (bottom left, verticallines). Hybridization of the recombinant versus rucuca will produce tagdifferences wherever the recombinant chromosome contains FRT 82 material(bottom right, vertical lines).

FIG. 3 illustrates that SNP detection is additive with differentrestriction enzymes. FIG. 3A: Experiments digesting the same genomic DNAwith different restriction enzymes will identify different SNPs in thegenome (mock array pictures). FIG. 3B: These SNPs can be combined tocreate a more detailed SNP map than would be possible using any singledigestion (SNPs identified with different restriction enzymes are shownwith thick and thin lines).

FIG. 4 illustrates mapping of bulk recombinants. FIG. 4A: Hybridizationof single recombinant lines versus rucuca line. Vertical lines indicateSNPs and FRT 82 chromosomal material. SNPs in common between the threerecombinants suggest mutation is in that region (dashed box). FIG. 4B:Isolating tags from all three lines and hybridizing simultaneouslyversus rucuca line creates a gradient of intensity ratios, with thehighest intensity in the common region (thick vertical bars).

FIG. 5 is a microarray, illustrating a test of a representativerestriction tag Protocol described in Example 1. Genomic DNA from FRT 82and rucuca was digested with BamHI after shearing and hybridized to agenomic array (one block shown). Strong “red” and “green” spots wereseen (representative ones of which are indicated with arrows),suggesting SNPs created differential tag presence.

FIG. 6 is a schematic of a representative protocol to extend labelednucleotides from mismatched restriction sites. The extra cut site inIndividual 1 creates two DNA fragments that bind to a single longerfragment in Individual 2 (step 2, center). One of these shorterfragments acts as a primer for extension along the longer fragmenttemplate (from Individual 2). Fluorescently-labeled nucleotides areincorporated, allowing detection on, for instance, a microarray.

FIG. 7 illustrates testing and demonstration of an extension method fordetection of polymorphisms. FIG. 7A: Test plasmids were cut at commonXhoI sites (lines) and the plasmid at right cut at a BamHI site(triangle). FIG. 7B: Annealing of plasmids expected to form a perfectmatch (left) and a mismatch of two shorter fragments onto a longerfragment (right). FIG. 7C: Fluorescently labeled nucleotides wereincorporated and material hybridized to test array of larger XhoIfragment (columns 1, 3, and 5) and shorter fragment (columns 2, 4, and6). Fluorescent material was only seen hybridizing to larger fragmentspot. FIG. 7D: Test of protocol after annealing FRT 82 and rucucagenomes digested by BamHI. Red spots (indicated with arrows) are seen ongenomic array at a rate similar to predictions.

FIG. 8 illustrates mapping of a recombination breakpoint. Genomic DNAfrom a recombinant line (thick portion of the line is rucuca DNA, thinis FRT 82 DNA) was annealed to FRT 82 parental DNA after digestion.Extension from mismatched cut sites results in strong labeling of spotsafter position 14,600,000. Labeling observed to the left of thisposition is at known repetitive DNA that is labeled in all experiments.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. In at least some cases, only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand.

SEQ ID NOs: 1 and 2 are the nucleic acid sequences of syntheticoligonucleotides used to generate a representative biotin linkerspecific for an EcoRI digestion.

SEQ ID NOs: 3 and 4 are the nucleic acid sequences of oligonucleotidesused to generate blunt-end linkers for use with random amplification.

DETAILED DESCRIPTION I. Abbreviations

cDNA: complementary DNA

DNA: deoxyribonucleic acid

EST: expressed sequence tag

GRIdS: genome-wide RFLP identification and segregation

PCR: polymerase chain reaction

RAD: restriction site associated DNA

RE: restriction enzyme (endonuclease)

RNA: ribonucleic acid

QTL: quantitative trait locus mapping

SNP: single nucleotide polymorphism

SNV: single nucleotide variant

ssRNA: single stranded RNA

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Addressable: Capable of being reliably and consistently located andidentified, as in an addressable location on an array.

Array: An arrangement of molecules, particularly biologicalmacromolecules (such as polypeptides or nucleic acids) in addressablelocations on a substrate, usually a flat substrate such as a membrane,plate or slide. The array may be regular (arranged in uniform rows andcolumns, for instance) or irregular. The number of addressable locationson the array can vary, for example from a few (such as three) to morethan 50, 100, 200, 500, 1000, 10,000, or more. A “microarray” is anarray that is miniaturized to such an extent that it benefits frommicroscopic examination for evaluation.

Within an array, each arrayed molecule (e.g., polynucleotide oroligonucleotide) is addressable, in that its location can be reliablyand consistently determined within the at least two dimensions, usuallydefined and on by the array surface. Thus, in ordered arrays thelocation of each molecule sample (feature, element) is usually assignedto the sample at the time when it is spotted onto or otherwise appliedto the array surface, and a key may be provided in order to correlateeach location with the appropriate feature. Often, ordered arrays arearranged in a symmetrical grid pattern, but samples could be arranged inother patterns (e.g., in radially distributed lines, spiral lines, orordered clusters).

Arrays are usually computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation (such as identification of the arrayed sample andhybridization or binding data, including for instance one or more signalintensity readings). In some examples of computer readable arrayformats, the individual spots on the array surface will be arrangedregularly, for instance in a Cartesian grid pattern, that can becorrelated to address information by a computer.

The sample application spot (or feature, or element) on an array mayassume many different shapes. Thus, though the term “spot” may be usedherein, it refers generally to a localized deposit of nucleic acid orother biomolecule, and is not limited to a round or substantially roundregion. For instance, substantially square regions of application can beused with arrays, as can be regions that are substantially rectangular(such as a slot blot-type application), triangular, oval, irregular, andso forth. The shape of the array substrate itself is also immaterial,though it is usually substantially flat and may be rectangular or squarein general shape.

Binding or interaction: An association between two substances ormolecules, such as the hybridization of one nucleic acid molecule toanother (or itself). The disclosed oligonucleotide arrays are used todetect binding of a labeled nucleic acid molecule (target) to animmobilized nucleic acid molecule (probe) in one or more features of thearray. A labeled nucleic acid molecule “binds” to a nucleic acidmolecule in a spot on an array if, after incubation of the (labeled)target molecule (usually in solution or suspension) with or on the arrayfor a period of time (usually 5 minutes or more, for instance 10minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes ormore, for instance over night or even 24 hours), a detectable amount ofthat molecule associates with a nucleic acid feature of the array tosuch an extent that it is not removed by being washed with a relativelylow stringency buffer (e.g., higher salt (such as 3×SSC or higher), roomtemperature washes). Washing can be carried out, for instance, at roomtemperature, but other temperatures (either higher or lower) also can beused.

Targets will bind probe nucleic acid molecules within different featureson the array to different extents, based at least on sequence homology,and the term “bind” encompasses both relatively weak and relativelystrong interactions. Thus, some binding will persist after the array iswashed in a more stringent buffer (e.g., lower salt (such as about 0.5to about 1.5×SSC), 55-65° C. washes).

Where the probe and target molecules are both nucleic acids, binding ofthe test or reference molecule to a feature on the array can bediscussed in terms of the specific complementarity between the probe andthe target nucleic acids.

cDNA: A DNA molecule lacking internal, non-coding segments (introns) andregulatory sequences which determine transcription. cDNA may besynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Cell sample: A sample of cells, either which may be homogenous orheterogeneous as to cell type, from which nuclei can be harvested. Inparticular examples, cell samples are taken from different primarycells; from embryonic stem cells; an immortalized cell line; ahomologous primary cell sample; from a cell type at different stages ofdevelopment; from different times in a disease progression; frominfected and uninfected homologous cells; from cells (or cells fromsubjects) treated with different conditions (e.g., drugs, drug regimes,temperature or other grown characteristics, carbon sources or foodsupplies), and so forth.

DNA: DNA is a long chain polymer that contains the genetic material ofmost living organisms (the genes of some viruses are made of ribonucleicacid (RNA)). The repeating units in DNA polymers are four differentnucleotides, each of which includes one of the four bases (adenine,guanine, cytosine and thymine) bound to a deoxyribose sugar to which aphosphate group is attached. Triplets of nucleotides (referred to ascodons) code for each amino acid in a polypeptide, or for a stop signal.The term “codon” is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Enriched: The term “enriched” means that the concentration of a materialis at least about 2, 5, 10, 100, or 1000 times its natural concentration(for example), advantageously at least 0.01% by weight. Enrichedpreparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are alsocontemplated.

EST (Expressed Sequence Tag): A partial DNA or cDNA sequence, typicallyof between 200 and 2000 sequential nucleotides, obtained from a genomicor cDNA library, prepared from a selected cell, cell type, tissue ortissue type, organ or organism, which corresponds to an mRNA of a genefound in that library. An EST is generally a DNA molecule sequenced fromand shorter than the cDNA from which it is obtained.

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forexample Cy3, FITC, and Oregon Green, are characterized by their emissionat wavelengths generally in the range of 515-540λ. Red fluorophores, forexample Texas Red, Cy5 and tetramethylrhodamine, are characterized bytheir emission at wavelengths generally in the range of 590-690λ.

Examples of fluorophores that may be used are provided in U.S. Pat. No.5,866,366 to Nazarenko et al., and include for instance:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

Other contemplated fluorophores include GFP (green fluorescent protein),Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivativesthereof. Other fluorophores known to those skilled in the art may alsobe used.

Genomic DNA: The DNA found within the nucleus and containing anorganism's genome, which is passed on to its offspring as informationfor continued replication and/or propagation and/or survival of theorganism. The term can be used to distinguish between other types ofDNA, such as DNA found within plasmids or organelles.

High throughput genomics: Application of genomic or genetic data oranalysis techniques that use microarrays or other genomic technologiesto rapidly identify large numbers of genes or proteins, or distinguishtheir structure, expression or function from normal or abnormal cells ortissues. It is particularly contemplated that high throughput genomicsin some instances will include the detection or differentiation ofpolymorphisms between two individuals or reference samples, or betweenan individual and a reference sample.

Human Cells: Cells obtained from a member of the species Homo sapiens.The cells can be obtained from any source, for example peripheral blood,urine, saliva, tissue biopsy, surgical specimen, amniocentesis samplesand autopsy material. From these cells, genomic DNA, cDNA, mRNA, RNA,cRNA, and/or protein can be isolated or generated.

Hybridization: Nucleic acid molecules that are complementary to eachother hybridize by hydrogen bonding, which includes Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleotide units. For example, adenine and thymine are complementarynucleobases that pair through formation of hydrogen bonds.“Complementary” refers to sequence complementarity between twonucleotide units. For example, if a nucleotide unit at a certainposition of an oligonucleotide is capable of hydrogen bonding with anucleotide unit at the same position of a DNA or RNA molecule, then theoligonucleotides are complementary to each other at that position. Theoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotide units which can hydrogen bond with each other.

“Specifically hybridizable” and “complementary” are terms that indicatea sufficient degree of complementarity such that stable and specificbinding occurs between the oligonucleotide and the DNA or RNA or PNAtarget. An oligonucleotide need not be 100% complementary to its targetnucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, for example under physiological conditionsin the case of in vivo assays, or under conditions in which the assaysare performed.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989), chapters9 and 11, herein incorporated by reference.

In vitro amplification: Techniques that increase the number of copies ofa nucleic acid molecule in a sample or specimen. An example of in vitroamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid.

The product of in vitro amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques.

Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Label: Detectable marker or reporter molecules, many of which can beattached to nucleic acids. Typical labels include fluorophores,radioactive isotopes, ligands, chemiluminescent agents, metal sols andcolloids, and enzymes. Methods for labeling and guidance in the choiceof labels useful for various purposes are discussed, e.g., in Sambrooket al., in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989) and Ausubel et al., in Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Intersciences(1987).

Mutation: Any change of the DNA sequence within a gene or chromosome. Insome instances, a mutation will alter a characteristic or trait(phenotype), but this is not always the case. Types of mutations includebase substitution point mutations (e.g., transitions or transversions),deletions, and insertions. Missense mutations are those that introduce adifferent amino acid into the sequence of the encoded protein; nonsensemutations are those that introduce a new stop codon. In the case ofinsertions or deletions, mutations can be in-frame (not changing theframe of the overall sequence) or frame shift mutations, which mayresult in the misreading of a large number of codons (and often leads toabnormal termination of the encoded product due to the presence of astop codon in the alternative frame).

This term specifically encompasses variations that arise through somaticmutation, for instance those that are found only in disease cells, butnot constitutionally, in a given individual. Examples of suchsomatically-acquired variations include the point mutations thatfrequently result in altered function of various genes that are involvedin development of cancers. This term also encompasses DNA alterationsthat are present constitutionally, that alter the function of theencoded protein in a readily demonstrable manner, and that can beinherited by the children of an affected individual. In this respect,the term overlaps with “polymorphism,” as discussed below, but generallyrefers to the subset of constitutional alterations that have arisenwithin the past few generations in a kindred and that are not widelydisseminated in a population group. In particular embodiments, the termis directed to those constitutional alterations that have major impacton the health of individuals having the mutation.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompassing known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Nucleic acid array: An arrangement of nucleic acids (such as DNA or RNA)in assigned locations on a matrix, such as that found in cDNA arrays, orin the herein described genomic tag arrays.

Nucleic acid molecules representing genes: Any nucleic acid, for exampleDNA (intron or exon or both), cDNA or RNA, of any length suitable foruse as a probe or other indicator molecule, and that is informativeabout the corresponding gene. In particular embodiments, there areprovided genomic tags, generated through restriction enzyme digestion ofgenomic DNA, which are useful as nucleic acid molecules that representgenes, parts of genes, or polymorphisms or variants in genes or othergenomic sequences.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: A linear single-stranded polynucleotide sequenceranging in length from 2 to about 5,000 bases, for example apolynucleotide (such as DNA or RNA) which is at least 6 nucleotides, forexample at least 10, 12, 15, 18, 20, 25, 50, 100, 200, 1,000, or even5,000 nucleotides long. Oligonucleotides are often synthetic but canalso be produced from naturally occurring polynucleotides.

An oligonucleotide analog refers to moieties that function similarly tooligonucleotides but have non-naturally occurring portions. For example,oligonucleotide analogs can contain non-naturally occurring portions,such as altered sugar moieties or inter-sugar linkages, such as aphosphorothioate oligodeoxynucleotide. Functional analogs of naturallyoccurring polynucleotides can bind to RNA or DNA, and include peptidenucleic acid (PNA) molecules. Such analog molecules may also bind to orinteract with polypeptides or proteins.

Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbonecomprised of monomers coupled by amide (peptide) bonds, such as aminoacid monomers joined by peptide bonds.

Plant cells: Cells obtained from any member of the Plantae Kingdom, acategory which includes, for example, trees, flowering and non floweringplants, grasses, and Arabidopsis. The cells can be obtained from anypart of the plant, for example roots, leaves, stems, or any flower part.From these cells, nucleic acid (including DNA, RNA, and so forth) and/orprotein can be isolated.

Polymorphism: Variant in a sequence of a gene, or any genomic sequence,usually carried from one generation to another in a population.Polymorphisms can be those variations (nucleotide sequence differences)that, while having a different nucleotide sequence, produce functionallyequivalent gene products, such as those variations generally foundbetween individuals, different ethnic groups, and geographic locations.The term polymorphism also encompasses variations that produce geneproducts with altered function, i.e., variants in the gene sequence thatlead to gene products that are not functionally equivalent. This termalso encompasses variations that produce no gene product, an inactivegene product, a truncated gene product, or increased or increasedactivity gene product.

It is contemplated herein that polymorphisms may be both singlenucleotide changes (e.g., single nucleotide polymorphisms, SNPs) orshort deletions or insertions within a genomic sequence.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule or protein that islinked to the variation (e.g., an alteration of a secondary structuresuch as a stem-loop, or an alteration of the binding affinity of thenucleic acid for associated molecules, such as polymerases, RNAses, achange in the availability of a site for cleavage by a restrictionendonuclease, either the formation of a new site, or lose of a site, andso forth).

Processing (of a sample of nucleic acid): Any laboratory procedure thatis carried out to alter or affect a nucleic acid molecule or sample ofnucleic acids. This includes, for instance, digestion of the nucleicacid with a specific or non-specific nuclease, ligation of a one or morenucleotide bases, oligonucleotide(s), or polynucleotide (such as avector) (or both) to the nucleic acid, sequencing of a nucleic acid,modification of one or more bases within a nucleic acid (e.g., chemicalmodification), purification of a nucleic acid or collection thereof fromthe milieu in which it is (they are) found, otherwise altering thatmilieu (for instance, by changing the pH, temperature, time ofincubation, salt concentration, and so forth), or in any way affectingthe nucleic acid sample.

A nucleic acid sample (such as a DNA sample) that has been processed (inone or more ways, or using one or more specific procedures) can bereferred to as a processed sample of nucleic acids. More specifically,by way of example a nucleic acid sample or mixture of nucleic acids thathas been treated with a nuclease can be referred to as a nucleasedsample (e.g., a DNA sample may be referred to as a DNAsed sample afterit is treated with DNAse). A nucleic acid that has been “polished” sothat there are no overhanging nucleotides (either by remove of theoverhang, or filling in with a ligase or polymerase) can be referred toeither as polished or blunted, or “blunt-ended” (in reference to theblunt (non-overhanging) ends after the process.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nucleicacid preparation is one in which the specified protein is more enrichedthan the nucleic acid is in its generative environment, for instancewithin a cell or in a biochemical reaction chamber. A preparation ofsubstantially pure nucleic acid may be purified such that the desirednucleic acid represents at least 50% of the total nucleic acid contentof the preparation. In certain embodiments, a substantially pure nucleicacid will represent at least 60%, at least 70%, at least 80%, at least85%, at least 90%, or at least 95% or more of the total nucleic acidcontent of the preparation.

The term purified, in some embodiments, refers to the separation ofnucleic acids of certain sizes or size ranges from a mixture of, forinstance, fragmented longer nucleic acids. Thus, for instance, it isappropriate to refer to purifying from a preparation of genomic DNA acollection of nucleic acid fragments of a set range of length, such asabout 200 bp to about 500 bp, about 500 bp to about 1000 bp, about 750bp to about 1500 bp, about 1000 bp to about 2000 bp, about 1000 bp,about 1500 bp, about 2000 bp, about 3000 bp, and so forth. Preparationof relatively long sets of fragments (e.g., 1 kb or more, 2 kb or more,3 kb or more, 5 kb or more, and so forth) are also contemplated. In eachinstance, the reference to a purified preparation (e.g., a purifiedpreparation of genomic fragments of about 1 kb) does not requireabsolute purity with regard to the length of the molecules. Rather, asdiscussed above, this refers to a preparation wherein the specifiednucleic acid length represents the length of at least 50% of themolecules in a mixed preparation. In certain embodiments, asubstantially pure nucleic acid of a specified length will contain atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, or atleast 95% or more nucleic acid molecules of approximately the specifiedlength within the total nucleic acid content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence,or a sequence that has been generated through a naturally occurring orinduced genetic recombination event. Artificial recombinant combinationscan be accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques.

Recombinant line: A line or strain, or individual, set of individuals,etc., that differs from a parent thereof by recombination within itsgenome. Recombination is generally the process by which offspring derivea combination of genes different from that of either parent. By way ofexample, this can occur by crossing over. Representative examplesprovided herein are recombinant lines of Drosophila. In humans, childrenare ‘recombinant lines’ of the parents, in that the paternal andmaternal chromosomes are each combinations of the different grandparentchromosomes. Similarly, a field test of a crop will typically be a testof many different recombinants (each of which could be used to give riseto a recombinant line) derived from a set of parents. So in anysituation where there is a set of progeny, and some have a trait andsome do not, those two groups could be compared on an array such asthose described herein, for parental chromosome material in commonwithin a group and different between groups.

Regulatory sequences or elements: These terms refer generally to a classof DNA sequences that influence or control expression of genes. Includedin the term are promoters, enhancers, locus control regions, boundaryelements/insulators, silencers, Matrix attachment regions (also referredto as scaffold attachment regions), repressor, transcriptionalterminators, origins of replication, centromeres, and meioticrecombination hotspots. Promoters are sequences of DNA near the 5′ endof a gene that act as a binding site for RNA polymerase, and from whichtranscription is initiated. Enhancers are control elements that elevatethe level of transcription from a promoter, usually independently of theenhancer's orientation or distance from the promoter. Locus controlregions (LCRs) confer tissue-specific and temporally regulatedexpression to genes to which they are linked. LCRs functionindependently of their position in relation to the gene, but arecopy-number dependent. It is believed that they function to open thenucleosome structure, so other factors can bind to the DNA. LCRs mayalso affect replication timing and origin usage. Insulators (also knowas boundary elements) are DNA sequences that prevent the activation (orinactivation) of transcription of a gene, by blocking effects ofsurrounding chromatin. Silencers and repressors are control elementsthat suppress gene expression; they act on a gene independently of theirorientation or distance from the gene. Matrix attachment regions (MARs),also known as scaffold attachment regions, are sequences within DNA thatbind to the nuclear scaffold. They can affect transcription, possibly byseparating chromosomes into regulatory domains. It is believed that MARsmediate higher-order, looped structures within chromosomes.Transcriptional terminators are regions within the gene vicinity thatRNA polymerase is released from the template. Origins of replication areregions of the genome that, during DNA synthesis or replication phasesof cell division, begin the replication process of DNA. Meioticrecombination hotspots are regions of the genome that recombine morefrequently than the average during meiosis.

RNA: A typically linear polymer of ribonucleic acid monomers, linked byphosphodiester bonds. Naturally occurring RNA molecules fall into threeclasses, messenger (mRNA, which encodes proteins), ribosomal (rRNA,components of ribosomes), and transfer (tRNA, molecules responsible fortransferring amino acid monomers to the ribosome during proteinsynthesis). Total RNA refers to a heterogeneous mixture containing allthree types of RNA molecules.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of nucleic acid or aminoacid sequences will possess a relatively high degree of sequenceidentity when aligned using standard methods. This homology will be moresignificant when the orthologous proteins or nucleic acids are derivedfrom species which are more closely related (e.g., human and chimpanzeesequences), compared to species more distantly related (e.g., human andC. elegans sequences). Typically, orthologs are at least 50% identicalat the nucleotide level and at least 50% identical at the amino acidlevel when comparing human orthologous sequences.

Methods of alignment of sequences for comparison are well known. Variousprograms and alignment algorithms are described in: Smith & Waterman,Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443,1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988 ;Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al.Computer Appls. Biosci. 8, 155-65, 1992; and Pearson et al., Methyl.Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10,1990, presents a detailed consideration of sequence alignment methodsand homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.Each of these sources also provides a description of how to determinesequence identity using this program.

Homologous sequences are typically characterized by possession of atleast 60%, 70%, 75%, 80%, 90%, 95% or at least 98% sequence identitycounted over the full length alignment with a sequence using the NCBIBlast 2.0, gapped blastp set to default parameters. Queries searchedwith the blastn program are filtered with DUST (Hancock and Armstrong,Comput. Appl. Biosci. 10:67-70, 1994). It will be appreciated that thesesequence identity ranges are provided for guidance only; it is entirelypossible that strongly significant homologs could be obtained that falloutside of the ranges provided.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, for instance due tothe degeneracy of the genetic code. It is understood that changes innucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid sequences that all encode substantially the sameprotein.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions, as described under “specific hybridization.”

Single Nucleotide Polymorphism (SNP): A single base (nucleotide)difference in a DNA sequence among individuals in a population. SNPs canbe causative (actually involved in or influencing the condition or traitto which the SNP is linked) or associative (linked to but not having anydirect involvement in or influence on the condition or trait to whichthe SNP is linked).

Specific hybridization: Specific hybridization refers to the binding,duplexing, or hybridizing of a molecule only or substantially only to aparticular nucleotide sequence when that sequence is present in acomplex mixture (e.g. total cellular DNA or RNA). Specific hybridizationmay also occur under conditions of varying stringency.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989 ch. 9 and 11). By wayof illustration only, a hybridization experiment may be performed byhybridization of a DNA molecule to a target DNA molecule which has beenelectrophoresed in an agarose gel and transferred to a nitrocellulosemembrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), atechnique well known in the art and described in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,1989).

Traditional hybridization with a target nucleic acid molecule labeledwith [³²P]-dCTP is generally carried out in a solution of high ionicstrength such as 6×SSC at a temperature that is 20-25° C. below themelting temperature, T_(m), described below. For Southern hybridizationexperiments where the target DNA molecule on the Southern blot contains10 ng of DNA or more, hybridization is typically carried out for 6-8hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to10⁹ CPM/μg or greater). Following hybridization, the nitrocellulosefilter is washed to remove background hybridization. The washingconditions should be as stringent as possible to remove backgroundhybridization but to retain a specific hybridization signal.

The term T_(m) represents the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target sequence hybridize to the target sequence atequilibrium. Because the target sequences are generally present inexcess, at T_(m) 50% of the probes are occupied at equilibrium. TheT_(m) of such a hybrid molecule may be estimated from the followingequation (Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390,1962):

T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(% formamide)−(600/1)

where 1=the length of the hybrid in base pairs.

This equation is valid for concentrations of Na⁺ in the range of 0.01 Mto 0.4 M, and it is less accurate for calculations of T_(m) in solutionsof higher [Na⁺]. The equation is also primarily valid for DNAs whose G+Ccontent is in the range of 30% to 75%, and it applies to hybrids greaterthan 100 nucleotides in length (the behavior of oligonucleotide probesis described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

Thus, by way of example, for a 150 base pair DNA probe derived from acDNA (with a hypothetical % GC of 45%), a calculation of hybridizationconditions required to give particular stringencies may be made asfollows: For this example, it is assumed that the filter will be washedin 0.3×SSC solution following hybridization, thereby: [Na^(+]=0.045) M;% GC=45%; Formamide concentration=0; 1=150 base pairs;T_(m)=81.5−16.6(log₁₀[Na⁺])+(0.41×45)−(600/150); and so T_(m)=74.4° C.

The T_(m) of double-stranded DNA decreases by 1-1.5° C. with every 1%decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973).Therefore, for this given example, washing the filter in 0.3×SSC at59.4-64.4° C. will produce a stringency of hybridization equivalent to90%; that is, DNA molecules with more than 10% sequence variationrelative to the target cDNA will not hybridize. Alternatively, washingthe hybridized filter in 0.3×SSC at a temperature of 65.4-68.4° C. willyield a hybridization stringency of 94%; that is, DNA molecules withmore than 6% sequence variation relative to the target cDNA moleculewill not hybridize. The above example is given entirely by way oftheoretical illustration. It will be appreciated that otherhybridization techniques may be utilized and that variations inexperimental conditions will necessitate alternative calculations forstringency.

Stringent conditions may be defined as those under which DNA moleculeswith more than 25%, 15%, 10%, 6% or 2% sequence variation (also termed“mismatch”) will not hybridize. Stringent conditions are sequencedependent and are different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH. An example of stringent conditions is a salt concentration of atleast about 0.01 to 1.0 M Na ion concentration (or other salts) at pH7.0 to 8.3 and a temperature of at least about 30° C. for short probes(e.g. 10 to 50 nucleotides). Stringent conditions can also be achievedwith the addition of destabilizing agents such as formamide. Forexample, conditions of 5×SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mMEDTA, pH 7.4) and a temperature of 25-30° C. are suitable forallele-specific probe hybridizations.

The following is an exemplary set of hybridization conditions and is notmeant to be limiting:

Very High Stringency (detects sequences that share 90% identity)Hybridization: 5x SSC at 65° C. for 16 hours Wash twice: 2x SSC at roomtemperature (RT) for 15 minutes each Wash twice: 0.5x SSC at 65° C. for20 minutes each

High Stringency (detects sequences that share 80% identity or greater)Hybridization: 5x-6x SSC at 65° C.-70° C. for 16-20 hours Wash twice: 2xSSC at RT for 5-20 minutes each Wash twice: 1x SSC at 55° C.-70° C. for30 minutes each

Low Stringency (detects sequences that share greater than 50% identity)Hybridization: 6x SSC at RT to 55° C. for 16-20 hours Wash at leasttwice: 2x-3x SSC at RT to 55° C. for 20-30 minutes each.

A perfectly matched probe has a sequence perfectly complementary to aparticular target sequence. The test probe is typically perfectlycomplementary to a portion (subsequence) of the target sequence. Theterm “mismatch probe” refers to probes whose sequence is deliberatelyselected not to be perfectly complementary to a particular targetsequence.

Transcription levels can be quantitated absolutely or relatively.Absolute quantitation can be accomplished by inclusion of knownconcentrations of one or more target nucleic acids (for example controlnucleic acids or with a known amount the target nucleic acidsthemselves) and referencing the hybridization intensity of unknowns withthe known target nucleic acids (for example by generation of a standardcurve).

Subject: Living, multicellular, vertebrate organisms, a category thatincludes both human and veterinary subjects for example, mammals, birdsand primates.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety, whether or not such incorporation is madeat the point of citation. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

There is provided in a first embodiment a method of nucleotidepolymorphism (or variant) discovery, which method involves isolatingfragment tags near restriction sites from genomic (or other) DNA;hybridizing the tags from two individuals or samples to a nucleic acidarray (or other detection platform, such as a collection of beads loadedwith target nucleic acid molecules); and detecting differentialhybridization, where differential hybridization indicates a sequencedifference (nucleotide polymorphism) between the two individuals orsamples.

There is provided in a second embodiment a method of nucleotidepolymorphism (or variant) discovery, which method involves annealing toeach other restriction enzyme-digested genomic DNA fragments from twoindividuals or samples; adding components for primer extension, inpresence of a label; allowing extension along one or more differentiallycut genomic DNA fragments, which extension incorporates the label intothe extension products; and detecting labeled extension products, ifsuch are generated, where a labeled extension product is indicative of asequence difference (nucleotide polymorphism) between the twoindividuals or samples.

Yet other embodiments are collections of nucleic acids, particularlyarrays, such as nucleic acid arrays and microarrays, and bead libraries.One example of such is a restriction site tag array (or library), whichcomprises as features two or more genomic restriction fragment tags fromat least one individual. Another collection of nucleic acid moleculesprovided herein is a subtractive restriction site tag array (orlibrary), which includes as features two or more genomic restrictionfragment tags selected at least in part through subtractivehybridization of DNA from two individuals or samples in order to enrichthe feature set on the collection/array/library for genomic restrictionfragment tags that differ between the two individuals or samples.

Methods of use of nucleic acid collections, such as arrays and beadlibraries, as described herein are also provided. For instance,contemplated and described herein is use of restriction site tag arraysor subtractive restriction site tag arrays, for instance to detect anucleotide polymorphism between the genomes of two individuals or twosamples. Also described are hybridization methods for polymorphism (orvariant) identification, mapping and/or analysis, which methods comprisehybridizing a first and a second single-stranded nucleotide molecule(e.g., labeled nucleotide molecules) to a restriction site tag array ora subtractive restriction site tag array. By way of example,hybridization assays will in some instances include contacting at leastone labeled target nucleic acid sample with an array, such as arestriction site tag array or a subtractive restriction site tag array(or library), under conditions sufficient to produce a hybridizationpattern; and detecting the hybridization pattern.

In yet another embodiment the is provided a method that includesisolating fragment tags (for instance, tags of about 1 kb, though otherlengths are contemplated) near restriction sites from genomic or otherDNA; hybridizing the tags from two individuals or samples to acollection of nucleic acid elements (for instance, a nucleic acid arrayor a bead library); and detecting differential hybridization, wheredifferential hybridization indicates a sequence difference (nucleotidepolymorphism) between the two individuals or samples. By way of example,isolating fragment tags in some cases involves contacting a preparationof genomic or other DNA with a restriction endonuclease; subjecting thepreparation of genomic DNA shearing force to fragment the DNA; andisolating DNA fragments from the resulting preparation based at least inpart on their length, which are the fragment tags, wherein shearing anddigesting the genomic (or other) DNA with the restriction endonucleasecan occur in either order or concurrently. The collection of nucleicacid elements in various examples comprises genomic DNA fragments, cDNAsor fragments thereof, synthetic nucleic acid sequences, or a combinationthereof.

Also provided is a method that involves shearing genomic (or other) DNAto fragments about 3-5 kb in length (though other length ranges could beselected); filling in any resultant overhanging ends on the fragments;digesting the sheared (and back filled) DNA with a restriction enzyme,to produce a collection of DNA fragments at least some of which have arestriction site at one end and a blunt end on the other; isolating fromthe collection DNA fragments about 1 kb in length (though longer andshorter fragment sets are contemplated); ligating a linker to thesheared DNA at the restriction site to produce linkered DNA fragments;isolating Tinkered fragments; and recovering the DNA fragments bydigesting away the linker. These fragments are indicative of thesequence of the starting nucleic acids, for instance with regard topolymorphisms or other variants, due to differential enzyme digestion.

Also provided are restriction site tag arrays and bead libraries,comprising as features of the array or as nucleic acid components ofmembers of the bead library two or more (or a much larger number)genomic restriction fragment tags from at least one individual.

Methods of generating restriction site tag arrays or bead libraries (orother collects of restriction site tags) are described. In examples ofsuch methods, a collection of genomic DNA fragments is produced from anindividual, at least some fragments of which have a restriction site atone end and blunt at the other by, in either order or concurrently:shearing a preparation of genomic DNA from the individual to fragmentsabout 3-5 kb in length; and digesting the preparation of genomic DNAwith a restriction enzyme. From the collection of genomic DNA fragments,a subset of DNA fragments about 1 kb in length are isolated, therebyproducing a tag set; optionally fragments of the tag set are amplified.The fragments of the tag set (which have optionally been amplified) arethen used as features on the array or in the bead library, or moregenerally as members in the collection of nucleic acid molecules.

Subtractive restriction site tag arrays and bead library are alsoprovided, comprising as features of the array or as nucleic acidcomponents of members of the bead library two or more genomicrestriction fragment tags selected at least in part through subtractivehybridization of DNA from two individuals or samples in order to enrichthe feature set on the array or in the bead library for genomicrestriction fragment tags that differ between the two individuals orsamples. By way of example, such subtractive arrays/libraries areproduced using a method that involves first producing a collection ofgenomic DNA fragments from each of the two individuals or samples, atleast some fragments of which have a restriction site at one end andblunt at the other by, in either order or concurrently: shearing apreparation of genomic DNA from each individual to fragments about 3-5kb in length; and digesting each preparation of genomic DNA with arestriction enzyme. DNA fragments about 1 kb in length are isolated,thereby producing a tag set from each individual or sample, andsubtractive hybridization is performed between the two tag sets, toproduce a collection of genomic restriction fragment tags that differbetween the two individuals or samples (differential fragment tags). Thecollection of genomic restriction fragment tags (which optionally can beamplified) are then used as features on the array or in the beadlibrary.

Methods of using arrays, bead libraries, and other nucleic acid (e.g.,fragment tag or tag set) collections are also provided, includingmethods for detecting a nucleotide difference (or similarity) betweenthe genomes of two individuals or two samples.

Hybridization methods for polymorphism identification or analysis arealso provided, which involve hybridizing a first and a secondsingle-stranded nucleotide molecule, or a first and second mixture ofsingle-stranded nucleotide molecules, to an array or bead library (orother collection of nucleic acids) described herein, and detecting thepresence of differential hybridization. In an example hybridizationassay, steps include contacting at least one labeled target nucleic acidsample with an array or bead library (or other collection) underconditions sufficient to produce a hybridization pattern; and detectingthe hybridization pattern.

There is also provided a method, comprising annealing to each otherrestriction enzyme-digested genomic DNA fragments from two individualsor samples; adding components for primer extension, in presence of alabel capable of being integrated into resultant extension products;allowing extension along one or more differentially cut genomic DNAfragments, which extension incorporates the label into the extensionproducts; and detecting labeled extension products, if such aregenerated, where a labeled extension product is indicative of a sequencedifference (for instance, a nucleotide polymorphism) between the twoindividuals or samples.

Yet additional embodiments are methods that involve identifying one ormore genomic tags as polymorphic between two individuals or populationsusing any one of the methods described herein; identifying a tag ofinterest by selecting a polymorphic spot mapping within the chromosomalregion of interest and showing the presence of a tag in the populationnot having the trait of interest. designing amplification primers, oneof which is specific for the sequence of the tag of interest, the othercorresponding to sequence in a DNA adapter sequence (e.g., a relativelyshort nucleic acid, such as a linker, part of which complements theoverhanging nucleotide sequence of the restriction site used ingenerating the tag and which includes a sequence to which anamplification primer can bind) ligated to the restriction site,isolating tags from a bulk population that exhibits the trait ofinterest; and amplifying the isolated tags using the amplificationprimers.

IV. Methods for Detection of Polymorphisms

Described herein are methods for SNP (or other sequence variation)discovery and mapping and genotyping. These methods are substantiallybased on commonly used molecular biology protocols, do not require priorknowledge of the nucleic acid population (e.g., genome) sequence to beanalyzed, can be used to detect SNPs in any number of differentindividuals, and scale well so that increased time and effort yield moreSNPs and more genotypes. The provided methods make mapping mutationsfrom genetic screens more routine, and are expected to spur thedevelopment of technologies for association studies of disease mappingand other more complex applications in humans. In overview, the providedpolymorphism detection methods exploit differential restrictionendonuclease digestion, which is detected using various means, in orderto identify the presence of polymorphisms. Detection methods include,for instance, detection of differential hybridization due todifferential digestion patterns, with or without primer extension todifferentially label sub-populations of fragments.

A first method involves use of a restriction enzyme to cleave a targetDNA sequence, for instance into size limited (e.g., ˜1 kb, though otherlengths are contemplated) fragments; these fragments are referred toherein as tags, genomic tags, or genomic restriction tags. Optionally, asubset of all of the fragments generated by digesting a sheared orotherwise fragmented genomic preparation can be selected (e.g.,purified) for use in these methods. In individuals where a nucleotidepolymorphism disrupts a recognition sequence of the restrictionendonuclease, the associated tag will be absent. Alternatively, lookedat in a different way, a polymorphism may generate a new recognitionsequence for the restriction endonuclease, thereby generating a newfragment or removing a fragment because the original fragment is now tooshort to be selected. Comparison of digested DNA from two samples (e.g.,two individuals) can be used to detect polymorphisms. For instance,labeled tags from two samples are hybridized to an array, and thepresence of polymorphisms is detected as strong differentialhybridization of the labeled DNA fragments on array elements.

A second method identifies polymorphisms by annealing restrictionenzyme-digested genomic DNA from two individuals to each other. Ifnucleotide polymorphisms disrupt the restriction site in one of the twoindividuals, then the site of polymorphism will generate two shortfragments (from one individual) bound to a longer (undercut) singlefragment (from the other individual). A similar situation pertains if apolymorphism generates a new restriction enzyme recognition site, aswill be apparent. One of the short fragments annealed to the longerfragment from the other individual is able to act as a primer forextension by DNA polymerase, allowing the incorporation of a label(e.g., fluorescent dye-linked nucleotides) near the polymorphism site.Hybridization of labeled DNA to microarrays allows polymorphismdetection and discovery at the array elements with strong hybridizationsignal intensity, as these elements will have bound the newlysynthesized sequences that correspond to the sequence containing thepolymorphism.

Though not essential for analysis of the tag fragments generated by theprovided methods, there are also provided tag-specific arrays (referredto, for instance, as genomic tag arrays), and tag-specific beadlibraries, that can be beneficial in the analysis and detection ofpolymorphism. A genomic tag array or bead library consists of acollection of genomic DNA sequences flanking sites of digestion for aparticular restriction enzyme in the genome. It is recognized that inmost situations, such an array or library will contain an undersamplingof all possible tags. For example, there may be 500,000 to 1 millionEcoRI sites in the human genome; digestion would generate twice thatnumber of tags (one arising from either side of the cut). Often, only amaximum of about 1/10^(th) of these would be placed on an array (thoughhigher numbers are contemplated); thus, only a small percentage of tagswould have its partner flanking tag (from directly opposite therestriction digestion) also on the same array.

For subtractive arrays and bead libraries as discussed below, very largearrays or collections of beads would contain a higher percentage ofpaired flanking tags. While the presence of such redundancy would leadto more confidence in interpreting a signal at a particular SNP locus,only one of the tags needs to be present to assay any single location.When the experimental and array (or bead library) restriction sitesmatch, each element is capable of detecting a restriction site tag, oran extension from restriction site polymorphism. Such an array (or beadlibrary) contains a number of elements that are in common between thegenomes of interest and other elements unique to a genome.

Yet another type of array provided herein is a very efficient array thatcontains only (or substantially only) elements that differ betweenindividuals. This is generated using the tags that remain after the tagsfrom the two individuals undergo a round of subtractive hybridization, aprocedure that removes DNA in common. In such a subtractive genomic tagarray, a 5000 element array could assay 5000 polymorphism differencesbetween individuals.

The various approaches described herein allow SNPs between any twoindividuals (or any two samples, such as an individual and a referencesample) to be rapidly detected at higher numbers than would be possiblefrom all but the most specialized efforts available prior to thisteaching. The described approaches are flexible enough to be used in anorganism for which the genome sequence is not yet available, allowingSNP mapping of traits in a variety of organisms. Furthermore, they allowthe establishment of SNP maps for laboratory lines or naturalpopulations in which SNP alleles exist at different frequencies comparedto SNP maps already created. Thus, these techniques could impact avariety of efforts to map mutations or natural alleles that affectphenotypes or lead to disease.

Related techniques are described in U.S. patent application Ser. No.10/236,598, published as US2004/0048257, which is incorporated herein byreference in its entirety.

It will readily be appreciated that the methods (and arrays and othernucleic acid collections) described herein can be used with nucleicacids from any source or subject, including animals, plants, microbes,and fungi. Nucleic acid molecules that can be analyzed using disclosedmethods include any nucleic acid molecule capable of (or believed to)including at least one variant (e.g., polymorphism or mutation) whencompared to a reference or other sample. Though the provided methods arein some instances adapted for and explained in the context of genomicDNA (including chromosomal DNA), other nucleic acid molecules that canbe analyzed include, without limitation, plasmid DNA, human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), P1-derived artificial chromosomes (PACs),cosmids and fosmids. In particular contemplated examples, analyzednucleic acid molecules are genomic DNA. Genomic DNA may be obtained, forexample, from one or more cells by methods known to those of ordinaryskill in the art (for example, kits for this purpose are commerciallyavailable from Promega, Roche Biochemical, Bio-Nobile, BrinkmannInstruments, BIOLINE, MD Biosciences, and numerous other commercialsuppliers; see, also, Sambrook et al., Molecular Cloning: A LaboratoryManual, New York: Cold Spring Harbor Laboratory Press, 1989; Ausubel etal., Current Protocols in Molecular Biology, New York: John Wiley &Sons, 1998).

Genomic DNA can also be obtained from any biological sample that may beobtained directly or indirectly from a subject (e.g., a medical orveterinarian subject), including whole blood, plasma, serum, tears, bonemarrow, lung lavage, mucus, saliva, urine, pleural fluid, spinal fluid,gastric fluid, sweat, semen, vaginal secretion, sputum, fluid fromulcers and/or other surface eruptions, blisters, abscesses, and/orextracts of tissues, cells or organs. The biological sample may also bea laboratory research sample such as a cell culture supernatant. Sampleare collected or obtained using methods well known to those ordinarilyskilled in the art.

In specific examples, genomic DNA is eukaryotic genomic DNA. Genomic DNAcan be obtained from an organism (or cells thereof) for which thesequence of genomic DNA is substantially known, including for instance,human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus),zebrafish (Danio rerio), Caenorhabditis elegans, Drosophilamelanogaster, Anopheles gambiae, Arabidopsis thaliana, lotus (e.g.,Lotus corniculatus var. japonicus), tomato (e.g., Lycopersiconesculentum), soybean (Glycine max), corn (Zea mays), rice (e.g., Oryzasativa), or cottonwood (e.g., Populus spp.) genomic DNA. In addition,however, it is a particular benefit of the provided methods that thegenome sequence need not be know for the methods to provide polymorphismor variant information, including distinguishing individuals orpopulations, examples of which are provided.

The methods (arrays, etc.) provided herein can be used in the analysisof plant nucleic acids. Additional representative, non-limiting exampleplants include Arabidopsis; field crops (e.g. alfalfa, barley, bean,clover, corn, cotton, flax, lentils, maize, pea, rape/canola, rice, rye,safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetablecrops (e.g. asparagus, beet, brassica generally, broccoli, Brusselssprouts, cabbage, carrot, cauliflower, celery, cucumber (cucurbits),eggplant, lettuce, mustard, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.almond, apple, apricot, banana, blackberry, blueberry, cacao, cassaya,cherry, citrus, coconut, cranberry, date, hazelnut, grape, grapefruit,guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya,passion fruit, peach, peanut, pear, pineapple, pistachio, plum,raspberry, strawberry, tangerine, walnut, and watermelon); tree woodsand ornamentals (e.g. alder, ash, aspen, azalea, birch, boxwood,camellia, carnation, chrysanthemum, elm, fir, ivy, jasmine, juniper,oak, orchid, palm, poplar, pine, redwood, rhododendron, rose andrubber). By way of example, the polymorphism detection methods describedherein can be used to detect differences between (or similaritiesbetween) plants, including identifying related (or unrelated) plantspecimens.

Specific embodiments will be discussed more fully below, and in theExamples that follow.

V. Restriction Site Tags for Polymorphism Discovery and Mutation Mapping

Using restriction site tags for polymorphism discovery and mutationmapping is conceptually simple, and requires little prior knowledge ofthe organism's genome. The detection of SNPs and other polymorphismsdepends on the fact that a nucleotide polymorphism may (andstatistically will) disrupt the recognition sequence of a restrictionendonuclease (or generate a new recognition site). Cutting genomic DNAwith a restriction enzyme that recognizes, for instance, a 6-basepairsequence (a 6-cutter enzyme) will produce many thousands of genomicfragments, essentially all of which are flanked by the restriction site.A small region of DNA flanking the restriction site is then isolated, sothat the presence or absence of the site can be detected. By way ofexample, this system can be referred to as Genome-wide RFLPIdentification and Segregation (GRIdS). Alternatively, the resultant DNAtags can be referred to as RAD (Restriction site Associated DNA)markers, and the corresponding collections of tags as RAD arrays orlibraries.

One specific protocol employed to generate restriction site tags is asfollows (also see FIG. 1):

-   -   1) Shear genomic DNA to fragments 3-5 kb in length and fill in        ends.    -   2) Digest the sheared DNA with a restriction enzyme, producing a        collection of fragments at least some of which have a        restriction site at one end and a blunt end on the other.    -   3) Isolate DNA fragments 1 kb in length by, for instance, gel        purification.    -   4) Ligate a biotinylated linker to the sheared DNA at the        restriction site.    -   5) Isolate the biotinylated linker and attached DNA, thereby        isolating only those fragments that had at least one end with a        restriction site.    -   6) Recover the purified DNA fragments by digesting away the        linker.

The order of at least certain of these steps is not essential. Forinstance, shearing of the genomic DNA, though noted above to be a firststep, has successfully been carried out later in the procedure. By wayof specific example, shearing can be carried out after digestion withthe restriction enzyme. See, for instance, Example 1 below. It isbelieved that the order of digestion by an RE and shearing isimmaterial.

The final mixture now contains short tags from near the existingrestriction endonuclease cut sites. If the process is repeated on asecond individual, a similar collection of tags is generated. However,on one hand, any restriction site containing a polymorphism thatdestroys recognition by the selected RE in the second individual willnot create a fragment of DNA that can be ligated to a biotinylatedlinker, and thus no tags for that locus will be present in the purifiedDNA. If a polymorphism created a restriction site in the secondindividual where one did not exist in the first individual, then a tagassociated with the genomic DNA near that locus will be present in thesecond individual's tag mixture but not the first individual's mixture.

The presence of these RE tags can be detected in various ways, examplesof which are described herein. In a first embodiment, the differentialRE tags are detected, and optionally quantified, on a microarray bydifferential labeling (e.g., fluorescent labeling). For example, thegenomic tags from individual 1 could be labeled with Cy-3 and the tagsfrom individual 2 labeled with Cy-5. A spot on the array showing astrong signal in one channel but not the other would indicate that apolymorphism exists at that locus. Most array spots would have roughlyequal intensity, as most restriction sites would not be polymorphic. Thecreation of short tags around the restriction site is important to makethe absence of a restriction site detectable. If the two genomes weremerely digested and labeled, then all genomic DNA would be available forhybridization to the microarray, resulting in equal intensity signals atall array elements.

The frequency by which there will be a difference in tag representationdepends on the length of the restriction site sequence and the frequencyof nucleotide polymorphisms between the two individuals. An individualpolymorphic at a rate of 1 nucleotide in 100 will have an altered6-basepair restriction site once out of every 16 occurrences of thesite. This suggests a detectable tag difference could occur every 64 kb,if the restriction site occurs every 4 kb on average. A polymorphismrate of 1 in 500 would decrease the resolution to 320 kb. Forsimplicity, we will assume a polymorphism rate (single nucleotidepolymorphisms and small insertion or deletions) of 1 every 100nucleotides. For that rate, a genomic DNA microarray capable ofdetecting all tag differences in the Drosophila melanogaster genomewould identify ˜1800 SNPs in a single experiment.

There are many alternatives available for detecting tag differences, aswill be recognized by one of ordinary skill. In order of increasingability to detect tag differences, selected possibilities includesmall-scale sequencing, expression microarrays, restriction tagmicroarrays or other assayable nucleic acid collections (discussedbelow, in Section VII), and tiling path genomic microarrays.

The most common commercially available resource for genomics is anexpression microarray composed of random cDNAs that are PCR-amplifiedand spotted onto glass or another substrate. These are expected to allowdetection of tag differences at a reasonable scale. The Drosophilacommunity, for instance, has several options for expression arrays,including oligos in the 70-mer range, short PCR fragments from genomicprimers, and cDNA collections.

With expression arrays, tag differences far from the genomic location ofthe spotted material will not be detected, decreasing the resolution ofthe SNP map. For example, the Incyte primer set produces ˜15,000amplicons of 400-700 basepairs in length. The amplicons vary greatly indensity across the genome, but average one every 8,000 basepairs. Sinceonly restriction sites near an amplicon will create a tag capable ofhybridizing to the spotted material, only one out of every threeamplicons would detect a possible tag, and only one of every 50amplicons detect a tag difference, for a resolution of 400 kb betweendetectable tag differences. Spotted long oligos would decrease thisresolution, as the spotted target area would be decreased (70 basepairsversus 400-700 basepairs).

Arrays of full-length cDNAs would increase the resolution, except theyrarely represent the entire set of transcribed genes. However, fornon-sequenced genomes, this is likely to be the most common platform fordetection.

Arrays of genomic DNA provide a powerful platform for tag detection. Byway of example, a genomic tiling path microarray made up of the 3 kbsubclones used in sequencing the Drosophila melanogaster genome (Adamset al., Science 287(5461): 2185-95, 2000) has been generated for usewith methods described herein. This example array is composed of ˜65,000spots with greater than 70% coverage of the Drosophila genome. Most ofthe gaps in coverage are less than 1 kb and are not likely to causemissing data by the methods described herein. The use of sequencingsubclones allowed amplification of the different genomic inserts withthe same PCR primers, and thus inexpensively create a tool that hadpreviously only been available for much smaller genomes, such as yeast(Lieb et al., Nat Genet. 28(4): 327-34, 2001; Lee et al., Science298(5594): 799-804, 2002). The entire clone set has been roboticallypicked through to create an optimal overlapping path (each clone is halfcovered by the preceding clone and half covered by the next clone), withgreater coverage and a reduced number of spots.

For tag difference detection, the genomic array provides a nearlycomplete representation of restriction sites, and thus a possibleresolution of (4 kb*16 sites between disruptive SNPs), or 64 kb. Thisresolution may be difficult to achieve in practice, as a significantpercentage of 3 kb clones may include multiple restriction sites. Ifonly one of the sites contained a SNP, then the signal hybridized at thespot would only decrease by half or more, making the SNP calls lesscertain.

There is also described herein a simple method to create a powerfulmicroarray platform for mapping polymorphisms/SNPs (discussed in SectionVII, for instance). If the short tags from the above method were ligatedinto a plasmid, transformed into bacteria, then singly picked andamplified, each spot on the resulting array would contain a restrictionsite tag matching the labeled material. Thus, the resolution would scalewell with the number of sites picked, until repeat sampling ofpreviously picked sites begins to affect coverage.

One of the probable uses of routine polymorphism/SNP detection in modelorganisms is the mapping of mutations from genetic screens. The use ofSNP panels has greatly increased the speed of mapping, although only afew fly lines have been examined for SNPs. The methods described hereincan be used to create genomic tags in lines commonly used formutagenesis screens, such as the Drosophila lines FRT 82 and rucuca. TheFRT 82 line is typically mutagenized; mapping of the mutation is thenpartly accomplished by creating recombinants with the visible markerline rucuca. The recombination breakpoint can be roughly mapped by thepresence or absence of the visible markers.

Genomic DNA from the two Drosophila lines has been digested with therestriction enzyme BamHI. Although the choice of restriction enzyme issomewhat arbitrary, enzymes recognizing C-rich sites may detect moreSNPs as many SNPs are C to T transitions. The genomic tags from the twolines have been hybridized to a genomic microarray, to determine whicharray elements can detect SNPs differing between the two lines. By themicroarray assay described in Example 1, yellow array elements indicatea restriction site shared by both genomes, while red or green elementsindicate the presence of restriction sites unique to one of the genomes.

Genomic tags from several different recombinant lines can be collectedand hybridized to a microarray with, for instance, either the FRT 82 orthe rucuca parental line in the other fluorescent channel. Areas wherethe chromosomes match genetic material are expected to create onlyyellow spots, whereas areas matching the other parental line areexpected to have a pattern of tag differences identical to the initialarray of the two parentals. Thus, the breakpoint of recombination shouldbe where these two patterns switch modes, with hybridizations to eachparent producing opposite patterns (see FIG. 2). From such microarrayanalyses, it is possible to make specific predictions about where therecombination breakpoints fall along the chromosome. To confirm thesepredictions, the genome near the predicted breakpoint can be examined,and areas on either side in the parental lines and recombinant linessequenced to confirm the presence of SNPs disrupting restriction sites.

The number of polymorphisms detected goes up with additional experimentsusing different restriction endonucleases. Different restriction enzymeswould have a similar rate of SNP discovery, but different array elementswould show differential hybridization with each different restrictionendonuclease experiment. Thus, with repeated experiments, the totalnumber of detectable SNPs could approach the number of elements on thearray, with a correlated increase in mapping resolution (see FIG. 3).

By way of example, the creation of genomic tags using different 6-cutterrestriction enzymes can be examined. Each enzyme would query a differentset of possible SNPs in the genome, but yield essentially the samepredicted recombination breakpoint. Thus, combining experiments usingdifferent restriction enzymes should create a SNP map with a greatlyincreased number of testable genotypes compared to using a singleenzyme. It is proposed to use BamHI, NgoM IV, NotI and PvuI, thoughother 6-base cutters, or other combinations, will be apparent to thoseof ordinary skill in the art. Alternatively, 5-cutter restrictionenzymes (many of which are known to those of ordinary skill in the art)can be examined to determine if, within a specific system, a shorter tagsize is beneficial for discrimination of tag differences. Hybridizationof the tags to different array platforms is expected to yield similarbreakpoints, and each array platform can be evaluated for numbers ofSNPs detected and signal consistency.

One potentially powerful aspect of the polymorphism mapping strategiesdescribed herein is the capability to perform a mapping hybridization ofmultiple recombinant lines on a single array. Hybridizing tag fragmentsfrom a single recombinant line versus tags from a parental line willallow the recombination breakpoints to be mapped at high resolution, butmany recombinants would have to be hybridized to identify chromosomalregions in common and decrease the size of the region where the mutationis located.

If many recombinants are collected and have genomic tags isolated,either separately or in a single mixture, then the ratio of the signalsin the two fluorescent channels at any array element would reflect thefrequency in which that tag was present in the mixed population (seeFIG. 4). So, for example, in an array of FRT 82, where a mutation wasinduced, versus mixed recombinants containing the mutation, the areasurrounding the mutation will be present in all recombinants. As thedistance from the mutation increases, the proportion of recombinantlines containing FRT 82 material at that tag will decrease. Therefore,spots approaching the FRT 82 versus rucuca ratio will be the areaclosest to the mutation, and array elements on either side of that areashould show decreasing ratios, as more of the individuals contain rucucamaterial at that locus. Thus, identifying the likely region of amutation entails examining array spot ratios and finding the peak of thecurve. The resolution of the mapping is correlated with the number ofrecombinant lines tested, with a maximum resolution bound by the averagedistance between detectable tag differences, and the number of arrayelements. Bulk testing of recombinants can be examined by pooling thetags from, for instance, five recombinant lines tested earlier on anindividual basis and hybridizing the recombinant tag mixture to an arraywith the FRT 82 parental in the other channel, and another array withthe rucuca parental in the other channel.

VI. Extension from Mismatched Restriction Sites for PolymorphismDiscovery and Mapping

Another method is provide, which also depends on detection ofdifferential restriction enzyme digestion patterns; it thus shares someconceptual underpinnings with the method provided above. The procedureagain depends on the fact that a nucleotide polymorphism in arestriction enzyme cut site will prevent that site from being digestion.One representative specific embodiment of this procedure is as follows:

-   -   1) Digest the genomic DNA of two individuals or samples to be        compared with a restriction enzyme that recognizes a 6-basepair        sequence.    -   2) Isolate the digested DNA from each sample, then denature and        mix the digested DNA from the two samples together.    -   3) The DNA from an individual with a SNP will not be cut at a        particular site, but cut in the second individual (or vice        versa). Therefore, some of the DNA in the region of a        polymorphism will hybridize as two shorter fragments annealed to        a longer, uncut fragment.    -   4) Extend fluorescent-dye attached (or otherwise labeled)        nucleotides from one of the shorter fragments, using one        individual's longer, uncut fragment as template.    -   5) Apply the resultant DNA material to a microarray and detect        variation by identifying spotted material with high fluorescent        signal; high fluorescence is indicative of the presence of a        polymorphism.

As noted above, at least certain steps may be carried out in a differentorder during preparation of the tag fragments.

In this procedure, a polymorphism is detected by the presence of asignal on the array (see FIG. 6). Thus, a representative array analysiswould have sample from a recombinant line annealed to sample form aparental line in one fluorescent channel, and sample from the twoparentals annealed in another. The two parental lines annealed wouldproduce signal at all loci containing a polymorphism in one parent andnot the other. The recombinant annealed to one parental would produce asignal at all polymorphisms between the two as well; however, this wouldbe restricted to the chromosomal regions where the recombinant linecontains material from the other parent. Thus, the two parental linesannealed act as a source of SNP discovery, while the other fluorescentchannel (recombinant annealed to parental) provides a way to map theareas matching the annealed parental.

For detection of fluorescent-labeled material extended from restrictionsite mismatch, the microarray options such as those described above arepossible for this technique. The resolution of this approach is similarto the calculations of methods in Section V. A distinguishing feature isthat the approach above produced signal at every cut site, whereas thisapproach produces a signal only where a SNP in one genome disrupts a cutsite present in the other. Thus, multiple detected SNPs hybridizing to asingle array element are much less likely in this approach. This freesthe technique to be used with restriction enzymes cutting morefrequently. For example, a restriction enzyme recognizing afour-basepair site will cut on average every 256 basepairs. In themethod of Section V, this would create 12 independent sources ofhybridizable material for each 3 kb genomic clone spotted on an array.For the current method, only a small subset of the sites would contain apolymorphism and a source of fluorescence. For example, a polymorphismrate of 1 in 100 would modify one in twenty-five four cutter sites.Twenty-five 4-cutter sites would on average exist at every 6,400basepairs, so only half of the 3 kb genomic clones would contain asignal, and a smaller portion would have multiple signals.

It may be beneficial to compare this procedure to the proceduredescribed in Section V, by repeating the detection of SNPs between theFRT 82 and rucuca lines for the 6-cutter and 5-cutter restrictionenzymes previously used. The SNPs detected by one method can be comparedwith SNPs detected by the other. Loci containing probable SNPs by thismethod that were not scored by the above method can be isolated by PCRfrom, for instance, the parental and recombinant lines and sequenced todetermine which method correctly identified the SNP.

Because this procedure only creates a signal on the array when a SNP ispresent, it is feasible to use restriction enzymes that cut atfour-basepair recognition sites. Therefore, this class of enzymes can beused for SNP detection in the recombinant lines already used for the 5-and 6-cutter enzymes, described herein. It is noted that there aredifferent technical issues that may arise using the greaterSNP-detecting power of 4-cutter enzymes. For instance, the labeledfragment extends to the next restriction site by this method, so a6-cutter digestion will create several thousand nucleotides offluorescently-incorporated nucleotides, while a 4-cutter digestion isexpected to only label several hundred nucleotides. Thus, producing asignificantly intense hybridization to an array element may be moredifficult with 4-cutter fragments than with 6-cutter fragments.Optionally, additional measures can be taken to increase signalintensity. By way of example, amino allyl incorporation followed byfluorescent dye attachment may be used to achieve greater efficiency offluorescent dye incorporation. Alternatively, biotin-linked nucleotidescan be incorporated, followed by streptavidin amplification of dyeincorporation, to also increase fluorescence. The biotin could alsoallow purification of extension regions, which could be followed byaddition of linkers for, e.g., cRNA or PCR amplification (or otheranalysis or processing) of the regions of interest.

VII. Restriction Site Tag Microarrays and Other Detection Methods

As discussed in Section V above, there are many options available fordetection of tag differences using the differential RE tag detectionmethods or the detection of restriction site mismatch extension methodsdescribed herein. Most of these options are already available for anyorganism for which genomic approaches are used, such as cDNA arrays orarrays of long oligos. Arrays of restriction site tags are specificallyprovided herein. If a goal of a project is to map traits via SNPs, forinstance, then such an array make particular sense, as they allow forhigher resolution mapping than a microarray created for expressionstudies with the same number of array elements.

A genomic tag array consists of DNA flanking each site of digestion fora particular restriction enzyme in the genome. Thus, when theexperimental and array restriction sites match, each element of thearray is capable of detecting a restriction site tag, or an extensionfrom restriction site polymorphism. By way of example, a genomicrestriction site tag microarray can be generated using the followingprocedure:

-   -   1) Shear genomic DNA to fragments 3-5 kb in length.    -   2) Digest the sheared DNA with a restriction enzyme, producing a        fragment with a restriction site at one end and blunt on the        other.    -   3) Isolate DNA fragments 1 kb in length.    -   4) Ligate DNA fragments into a plasmid vector    -   5) Transform bacteria with plasmids.    -   6) Pick individual colonies.    -   7) Amplify inserts.    -   8) Spot onto array.

As noted above, certain steps may be carried out in a different orderthan is listed. For instance, shearing and restricting the genomic DNAmay be carried out in either order.

With regard to ligating the DNA fragments into the plasmid vector, itwill be appreciated that different cloning schemes could be used. By wayof example, it is specifically contemplated to digest the vector withthe original RE used to generate the fragments, plus a blunt cutter RE.In this procedure, the fragment will readily ligate to the non-bluntside, and the subsequent forced spatial localization of the blunt end tothe area around the vector will increase the ligation of that side.

Restriction tag arrays have several advantages for detectingpolymorphisms, such as SNPs. First, unlike spotted long oligonucleotideor cDNA arrays, this array would contain hybridizable material at everyelement when the array restriction enzyme used is matched with theexperimental restriction enzyme. A typical cDNA array element would onlyhave a ˜50% chance of containing DNA matching a particular tag, and along oligonucleotide array even less. Thus, the restriction tag arraywill maximize the number of polymorphisms detected.

For organisms with a genome where the sequence is not known, cDNA arrayshave been the only other option in the past. However, the genomic tagarray is more efficient in that there is less of a chance ofover-sampling one locus, compared to cDNA arrays that may have heavyrepresentation of a few highly transcribed genes. The genomic tag arrayalso samples the more highly polymorphic intergenic as well as generegion DNA, increasing the number of polymorphisms/SNPs detectedcompared to a cDNA array.

If SNPs are discovered, particularly using the procedures provided inSection V, then the length of the spotted elements on the analysis array(of whatever type) can be important. Array elements containing nucleicacid sequences that are too long will be hybridized to by multiplerestriction site tags, making interpretation of the results lesscertain. The average length of cDNAs or the 3 kb clones use herein forthe working example genomic array makes a 6-cutter enzyme most likely toobtain clear results. The shorter length of DNA spotted onto the genomictag array as described herein allows for restriction enzymes that cutmore frequently, such as those that recognize a 5 basepair sequence.Whereas in an individual polymorphic at a rate of 1 nucleotide in 100, aSNP altering a restriction site would occur every (4000 bp*100/6) or 66kb for a six-cutter enzyme, a five-cutter enzyme would be polymorphicevery (2000 bp*100/5) or 40 kb.

By way of example, a library of BamHI tags of 1 kb in length can beprepared from genomic DNA of the Drosophila melanogaster line FRT 82,commonly used for genetic screens. There are likely to be ˜30,000 BamHIsites in the genome. In a first instance, about 13,000 restriction sitetags could be amplified, for approximately ⅓ coverage of the genome, andthese elements spotted on glass slides. These procedures are routine tothose of ordinary skill in the art.

The genomic tag arrays can be employed in the protocols describedherein. The restriction site tag approach can be examined, for instance,by isolating BamHI tags from FRT 82 flies, rucuca flies (often used formapping by recombination), and a recombinant fly line containing amixture of rucuca and FRT 82 chromosomal material. One array ishybridized with FRT 82 tags versus rucuca tags; a second array ishybridized with FRT 82 tags versus recombinant tags, and a third arraywith rucuca tags versus recombinant tags. The first array allowsdiscovery of all detectable SNPs between the FRT 82 and rucucachromosomes. The second and third arrays should show tag differences inthe areas where the recombinant line does not match chromosomes materialwith the parental line being co-hybridized. These arrays are thereforeexpected to have a subset of SNPs compared to the first array, and thesecond and third should have complementary patterns of detectable tagdifferences. Because the genomic tag array will have a random set ofclones, spot identity is established after the experimental data iscollected, for instance by sequencing (e.g., pyrosequencing) of clonesof interest to place them in the genome.

Although a genomic tag array is optimized for experiments using genomictags created with the same restriction enzyme used to create the array,other restriction enzyme tags can also be used to increase the number ofSNPs detected. The mapping procedures can be repeated using tagsisolated with other restriction enzymes. By way of example, 6-, 5- and4-cutter enzymes can be used to create tags. The number of SNPs detectedwith each restriction enzyme for that specific genome can then bedetermined.

It is expected that 5-cutter tags will be present once per array element(where the array elements are about 1500 to 3000 bp), while 4-cuttertags will have multiple instances per element, and therefore complicateinterpretation of the results. In those instances where a fractionaldecrease in array element hybridization is consistently interpretable,then the use of 4-cutter enzymes will increase the resolution of thearray even more. Even without the use of 4-cutters, the ability to querydifferent SNPs with each restriction enzyme used is believed to allowfor maps of higher resolution, and provide independent confirmation ofpredicted regions containing mutations.

The genomic tag microarrays provided herein can map SNPs at a resolutionwhich is limited by the number of array elements on the microarray. Theresolution of a microarray with 10,000 unique tags can be no greaterthan 12 kb between detectable SNPs (the 120 million basepairs in theDrosophila genome divided by 10,000 elements, for instance). To achievethis resolution requires multiple procedures with different restrictionenzymes (for instance, twenty experiments involving 6-cutter enzymes,fewer experiments if 4- or 5-cutters are used). While this resolutionwould be adequate for mapping a mutation produced by a genetic screen inthe fly, this number of elements would produce lower resolution SNP mapsin organisms with larger genomes, although gene densities decrease asgenome size increases. Obviously, more elements could be added to thearray. However, most spotting array printers produce arrays with amaximum of 35,000 elements. Commercial arrays involving synthesis ofoligos in situ can produce several hundred thousand elements per array.

One low-cost and rapid method to map SNPs at a higher resolution wouldbe to do use a 10,000 element array to identify a region of interest,then produce a high resolution array of that smaller region. Forinstance, if the initial array analyses mapped a mutation to a 300 kbregion, then genomic tags could be isolated from BACs spanning thatregion. Only a few hundred tags would be needed to have 95% of all6-cutter sites from a single digest, so a reasonable strategy would beto isolate 200 tags each from two different restriction enzymedigestions. The creation of a mini-array of this type would take no morethan a few weeks and a few hundred dollars. The same material used toroughly map the mutation to that region could then be hybridized to themini-array to create a very high-resolution map of the region.

By way of example, a high-resolution genomic tag array of a 300 kbregion between ebony (e) and claret (ca), two visible markers on therucuca 3^(rd) chromosome, can be generated. Restriction tags generatedafter BamHI digestion of the two BACs that span this region (of whichthere are 384) can be generated and spotted onto glass slides. The 384tags are expected oversample most BamHI sites in the BACs, and 90% ofthe sites are expected to have at least one tag. Thus, the resolution ofSNPs detected should improve, as more sites in the region are detectableon the array. Genomic tags from recombinant lines thought to havebreakpoints in the region can be hybridized to this mini-array and theincrease in SNP detection assessed.

Non-array based strategies may be even more rapid at the scale ofdetecting tags from, for instance, a few hundred kilobases. At largerscales, the number of tags needed to be sequenced makes the costprohibitive. There are currently methods to purify genomic DNA from aparticular region, based on the hybridization of total genomic DNA tobead-bound BAC DNA (Bashiardes et al., Nature Methods 2:63-69, 2005).Thus, tags from the region of interest can be isolated, and individualtags sequenced. If particular tags were missing from one individual,then that would indicate the presence of a SNP at that site. Multipleoptions exist for rapid and/or low-cost sequencing, from pyrosequencingof individual tags (Ramon et al., J. Translat. Med. 1(9), p 1-10, Epub26 Nov. 2003), to SAGE-type strategies for sequencing of concatamers(Wang et al., Proc Natl Acad Sci USA 99(25):16156-61, 2002; Epub 2 Dec.2002); for a review of “fast” sequencing technologies, see Bonetta,Nature Methods 3(2):141-147, 2006. For projects where multiple regionsmay be of interest, such as mapping mutations from a genetic screen,rapid sequencing-based approaches may be most efficient. By way ofexample, genomic DNA of the two lines discussed above can be isolated bypurification on a single BAC, and pyrosequencing analysis applied to the384 tags from each line. Using these analyses, it can be determined iftag quantity in a particular situation can reliably indicate thepresence or absence of restriction sites in the recombinants.

It is further recognized that any short nucleotide tags associated withrestriction sites, as produced by the methods described, can be assayedor examined using platforms other than competitive hybridization tomicroarrays. By way of non-limiting example, two methods are describedfurther below: tag-specific amplification and bead-based hybridizationdetection. One of ordinary skill in the art will recognize thatadditional methods can be used, based on the teachings provided herein.For instance, high throughput analysis using a microfluidic system isanother option. By way of example, the following publications providedescriptions of various microfluidic systems useful for analysis ofbiomolecules including nucleic acids, and other systems will berecognized by those of ordinary skill in the art: Olsen et al., Anal.Chem. 74:1436-1441, 2002; Wei et al., Nucleic Acids Res. 33:e78, 2005;US 2005/0221373; US 2005/0053952; US 2005/0032076; US 2003/0032035; US2003/0165964; and US 2004/0110208

Once the presence of a tag has been determined as polymorphic betweentwo individual samples, one exemplary and non-limiting application ofthat information is to assay the presence (or absence) of that tag indifferent individuals or populations. A particular situation where thiswould be useful, for instance, is after a chromosomal region has beenidentified as potentially linked to a trait. Individual polymorphicmarkers within that region can then be assayed in bulk populationsamples to detect low frequency polymorphism states in the population.The presence of a tag inherited from a parent without the trait wouldallow that portion of the (potentially) linked region to be ruled out ascontaining the gene (more generally, sequence) of interest. Isolation ofthe DNA surrounding a restriction site allows this detection to becarried out by the following exemplary method:

-   -   1) Identify tags as polymorphic between two populations as        described herein (e.g., by array or other detection method).    -   2) Identify a tag of interest by selecting a polymorphic spot        mapping within the chromosomal region of interest and showing        the presence of a tag in the population not having the trait of        interest.    -   3) Design amplification primers, one of which is specific for        the sequence of the tag of interest, the other corresponding to        sequence in a DNA adapter sequence (e.g., a relatively short        nucleic acid, such as a linker, part of which complements the        overhanging nucleotide sequence of the restriction site used in        generating the tag and which includes a sequence to which an        amplification primer can bind) ligated to the restriction site.    -   4) Isolate tags from a bulk population that exhibits (at some        level) the trait of interest.    -   5) Amplify (e.g., using PCR or another amplification technique)        the isolated tags using the amplification primers.

If the tag of interest is amplified from the bulk population, itindicates that at least some individuals within that populationinherited DNA from a parent(s) without the trait, and therefore thetrait cannot be at the locus of the tag. By repeated sampling ofpotential tags of interest, the exact region always inherited from theparent with the trait can be determined.

The described PCR-based detection method results in an amplified DNAfragment of known size (known from the location of the restriction siteand the chosen primer site within the tag). The presence (or absence) ofamplified material can be detect by running the product of the PCRreaction through an agarose gel and visually inspecting it for a band ofthe predicted size, or by other art recognized methods.

Another representative method is a bead-based high-throughput system fordetecting polymorphic markers after tag isolation. PCR amplificationwithin oil droplets (emulsion PCR) on a bead-capture system has recentlybeen used to successfully create beads covered with millions of copiesof a short stretch of DNA (Margulies et al., Nature 437(7057):376-80,2005; Epub Jul. 31, 2005). See also U.S. Patent Publication2005/0079510, which describes methods for carrying out bead emulsionnucleic acid amplification to produce beads each of which is coveredwith a highly amplified sequence. Each of these beads can beconceptually treated as though it is an array element, with thedifference being that the DNA is not spotted on a solid glass slide orother contiguous surface, but instead the DNA is adhered to a bead inliquid suspension.

DNA from any source can be used for making a bead library—shearedgenomic DNA, the isolated tags or cDNAs, for example. The tags isolatedfrom two individuals or two populations, for instance, can be hybridizedto the bead suspension, after fluorescent labeling of tags (which allowstheir origin to be distinguished). As for read-out analysis by an arrayor microarray, there will be beads with no signal from either source oftags if the DNA amplified on the bead is not near a restriction site;there will be beads that have equal amounts of label (such asfluorescent label; Cy3 and Cy5 dyes, for example), suggesting that arestriction site is present in both tag sources in the DNA amplified onthat bead; and there will be beads with fluorescent label from a singlesource (Cy3 dye or Cy5 dye, for example), suggesting that the DNAamplified on the bead has a restriction site in one tag source but notthe other. Similarly, when assaying the presence of tags in a bulkedpopulation, there will be beads with varying relative amounts of Cy3 andCy5 dyes, indicating the relative proportion of individuals in thosepopulations with the particular polymorphic tag hybridized to the DNAcarried by the bead. An advantage of a bead-based system is that thenumber of possible tags assayed is not limited by the number of spots onthe array, and will most likely saturate all possible polymorphic tagsbetween the individuals or populations.

Beads with differential fluorescent intensities can be identified andselected (for instance, selected for further analysis) by a variety ofmethods, including sorting by fluorescence activated cell sorting (FACS)(Mastrobattista et al., Chem. Biol. 12(12): 1291-1300, 2005). Afterhybridization of tags to bead DNA, the hybridized beads can bere-packaged in oil droplets to allow sorting by FACS machine. Individualbeads are rapidly sorted by fluorescent signal into different capturebins by FACS, allowing the selection of beads with very high Cy3 or Cy5fluorescent signals (or any other differential fluorescence or other tagsignals).

Whereas an array-based system makes identification of individual arrayelements possible by tracking and/or sequencing the template of theindividual amplification reactions, a bead-based system requires beadDNA identification for every test. Although multiple methods arepossible, one example useful for such identification is sequencing ofhigh-intensity bead DNA for identification. After sorting by FACS, beadDNA can be diluted and PCR amplified for sequencing. See also publishedU.S. patent documents US 2002/0094116 and US 2006/0029267, for methodsof reading reporter labeled beads.

Once beads with differential signal have been sorted, DNA is releasedfrom the beads into individual containers (such as wells in a microtiterplate), and amplification and sequencing of individual samples can becarried out. The sequences that result would identify those tags thathad been selected by the FACS. The selected sequences could then bemapped to a known genomic sequence, or used to design primers foramplification of BAC libraries or other genomic resources, for instance.

If high numbers of bead identifications are desired, a serial analysisof gene expression (SAGE)-like approach can be utilized to sequencemultiple short representations of the DNA available in the selectedbeads. Beads can also be used as a source for array element generation,allowing the selection of highly polymorphic elements prior to laboriousamplification.

VIII. Subtracted Restriction Site Tag Microarrays and Libraries

As described above, genomic tag arrays and bead libraries consist of DNAflanking sites of digestion for a particular restriction enzyme in thegenome. A very efficient array (or set/library of nucleic acid-loadedbeads) would contain only elements that differ between individuals. Thisis possible if the tags from the two individuals undergo a round ofsubtractive hybridization, a procedure that removes DNA in common. Thus,a 5000 element array (or other collection of nucleic acid elements)could assay 5000 SNP differences between individuals. The creation of agenomic restriction site tag microarray would use the followingprocedure:

-   -   1) Shear genomic DNA to fragments 3-5 kb in length.    -   2) Digest the sheared DNA with a restriction enzyme, producing a        fragment with a restriction site at one end and blunt on the        other.    -   3) Isolate DNA fragments 1 kb in length.    -   4) Perform subtractive hybridization between the two tag sets.    -   5) Ligate DNA fragments into a plasmid vector.    -   6) Transform bacteria with plasmids.    -   7) Pick individual colonies.    -   8) Amplify inserts.    -   9) Spot onto array.

As noted with other methods described herein, certain steps may becarried out in a different order than is listed. For instance, shearingand restricting the genomic DNA may be carried out in either order.

Subtracted tag arrays can be used, for instance, in the ways describedabove for use with non-subtracted genomic tag arrays. By way of example,subtractive hybridization arrays can be generated for any particularspecies. In one particular embodiment, subtractive arrays can begenerated from oceanic and lake forms of stickleback. Such an arraycould then be used to map differences between these sticklebackvariants, for instance to map armor loss differences between thesepopulations.

Similarly, subtracted tag bead libraries and otherwise formattedcollections of subtracted nucleic acids can be produced and used.

IX. Computer Assisted (Automated) Detection and Analysis

The data generated by assaying the disclosed arrays or other collectionsof nucleic acids can be analyzed using known computerized systems. Forinstance, an array or collection can be read by a computerized “reader”or scanner adapted for the analysis of the format of the array orcollection, and quantification of the binding of target to individualaddresses (features) carried out using computer algorithms. Likewise,where a control target has been used, computer algorithms can be used tonormalize the hybridization signals in the different features. Suchanalyses can be referred to as “automated detection,” in that the datais being gathered by an automated reader system.

In the case of labels that emit detectable electromagnetic wave orparticles, the emitted light (e.g., fluorescence or luminescence) orradioactivity can be detected by very sensitive cameras, confocalscanners, image analysis devices, radioactive film or a phosphorimager,which capture the signals (such as a color image) from the array (orother signal source). A computer with image analysis software detectsthis image, and analyzes the intensity of the signal for each probelocation in the array. Signals can be compared between spots on a singlearray, or between arrays (such as a single array that is sequentiallyinterrogated with multiple different target molecule preparations), orbetween the labels of different targets (or combinations of targets) ona single array.

Computer algorithms can also be used for comparison between spots on asingle array or on multiple arrays. In addition, the data from an arraycan be stored in a computer readable form.

Certain examples of automated array readers (scanners) will becontrolled by a computer and software programmed to direct theindividual components of the reader (e.g., mechanical components such asmotors, analysis components such as signal interpretation and backgroundsubtraction). Optionally software may also be provided to control agraphic user interface and one or more systems for sorting,categorizing, storing, analyzing, or otherwise processing the dataoutput of the reader.

To “read” an array, an array that has been assayed with a detectabletarget to produce binding (e.g., a binding pattern) can be placed into(or onto, or below, etc., depending on the location of the detectorsystem) the reader and a detectable signal indicative of target bindingdetected by the reader. Those addresses at which the target has bound toan immobilized nucleic acid mixture provide a detectable signal, e.g.,in the form of electromagnetic radiation. These detectable signals couldbe associated with an address identifier signal, identifying the site ofthe “positive” hybridized spot. The reader gathers information from eachof the addresses, associates it with the address identifier signal, andrecognizes addresses with a detectable signal as distinct from those notproducing such a signal. Certain readers are also capable of detectingintermediate levels of signal, between no signal at all and a highsignal, such that quantification of signals at individual addresses isenabled.

Certain readers that can be used to collect data from the arrays,especially those that have been interrogated using a fluorescentlytagged molecule, will include a light source for optical radiationemission. The wavelength of the excitation light will usually be in theUV or visible range, but in some situations may be extended into theinfra-red range. A beam splitter can direct the reader-emittedexcitation beam into the object lens, which for instance may be mountedsuch that it can move in the x, y and z directions in relation to thesurface of the array substrate. The objective lens focuses theexcitation light onto the array, and more particularly onto the(polypeptide) targets on the array. Light at longer wavelengths than theexcitation light is emitted from addresses on the array that containfluorescently labeled target molecules (i.e., those addresses containinga nucleic acid molecule within a spot containing a nucleic acid moleculeto which the target binds).

In certain embodiments, the array may be movably disposed within thereader as it is being read, such that the array itself moves (forinstance, rotates) while the reader detects information from eachaddress. Alternatively, the array may be stationary within the readerwhile the reader detection system moves across or above or around thearray to detect information from the addresses of the array. Specificmovable-format array readers are known and described, for instance inU.S. Pat. No. 5,922,617, hereby incorporated in its entirety byreference. Examples of methods for generating optical data storagefocusing and tracking signals are also known (see, for example, U.S.Pat. No. 5,461,599, hereby incorporated in its entirety by reference).

For the electronics and computer control, a detector (e.g., aphotomultiplier tube, avalanche detector, Si diode, or other detectorhaving a high quantum efficiency and low noise) converts the opticalradiation into an electronic signal. An op-amp first amplifies thedetected signal and then an analog-to-digital converter digitizes thesignal into binary numbers, which are then collected by a computer.

In embodiments that employ bead-based collections of nucleic acids forread-out, beads with differential fluorescent intensities can beidentified and selected by a variety of methods, including sorting byfluorescence activated cell sorting (FACS) (Mastrobattista et al., ChemBiol. 12(12):1291-1300, 2005). See also published U.S. patent documentsUS 2002/0094116 and US 2006/0029267, for methods of reading reporterlabeled beads.

X. Polymorphism Detection Kits

Polymorphism (or variant) detection arrays or collections of tags (forinstance, tag sequences immobilized on beads or other non-array basedcollections) as disclosed herein can be supplied in the form of a kitfor use in nucleic acid analyses. In such a kit, at least one array (orother collection) is provided. The kit also includes instructions,usually written instructions, to assist the user in probing the array orcollection. Such instructions can optionally be provided on a computerreadable medium.

Kits may additionally include one or more buffers for use during assayof the provided array or collection. For instance, such buffers mayinclude a low stringency wash, a high stringency wash, and/or astripping solution useful with array analysis. These buffers may beprovided in bulk, where each container of buffer is large enough to holdsufficient buffer for several probing or washing or strippingprocedures. Alternatively, the buffers can be provided in pre-measuredaliquots, which would be tailored to the size and style of arrayincluded in the kit. Kits for use with bead-based collections also maybe provided with reagents or buffers useful in carrying outhybridization reactions on the beads, as well as components useful inanalysis or “reading” of the hybridized beads.

Certain kits may also provide one or more containers in which to carryout array-assaying reactions.

Kits may in addition include either labeled or unlabeled control targetmolecules. Such molecules can be provided to provide for internal testsof the labeling procedure or interrogation of the array or other nucleicacid collection, or both. The control target molecules may be providedsuspended in an aqueous solution or as a freeze-dried or lyophilizedpowder, for instance. The container(s) in which the controls aresupplied can be any conventional container that is capable of holdingthe supplied form, for instance, microfuge tubes, ampoules, or bottles.In some applications, control probes may be provided in pre-measuredsingle use amounts in individual, typically disposable, tubes, orequivalent containers.

Specific examples of target molecules include preparations of nucleicacids that correspond to the sequences used to construct the array ornon-array based collection provided in the kit. Other control targetsmay be targets that comprise one or more previously identified variantsfor the sequence(s), such that the user is provided with a non-consensuscontrol “test” sample.

The amount of each control target supplied in the kit can be anyparticular amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, sufficient control target(s) likely will be provided toperform several controlled analyses of the array. Likewise, wheremultiple control targets are provided in one kit, the specific targetsprovided will be tailored to the market and the accompanying kit.

In some embodiments, kits may also include the reagents necessary tocarry out one or more target-labeling reactions. The specific reagentsincluded will be chosen in order to satisfy the end user's needs,depending on the type of target molecule (e.g., DNA or RNA or PNA orsome other derivative) and the method of labeling (e.g., radiolabelincorporated during target synthesis, attachable fluorescent tag,element of a strept/avidin:biotin system, etc.).

Further kits are provided for the labeling of target molecules for usein assaying arrays or provided herein. Such kits may optionally includean array or collection to be assayed by the so labeled target molecules.Other components of the kit are largely as described above for kits forthe assaying of arrays and other collections.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 Use of Restriction Site Tags for Single NucleotidePolymorphism (SNP) Discovery and Mutation Mapping

This example provides representative methods for generating restrictionsite tags and their use in detection of polymorphisms. A schematicoverview of the exemplary method is provided in FIG. 1.

Methods Digestion of Genomic DNA

Genomic DNA was prepared using standard techniques. (Since the DNApreparation is to be sheared, even relatively poor or degraded genomicsamples are satisfactory.) The genomic DNA preparation was then cut witha 6-cutter restriction endonuclease, EcoRI in the current instance,though others are contemplated. 10 μg of clean, non-degraded genomic DNAfrom rucuca and FRT82 flies was brought to 175 μl with dH₂O. To this, 20μl of 10×6-cutter buffer (in this case, EcoRI buffer) was added and thetube flicked gently to mix. The reaction mixture was brought to thebottom of the tube by briefly spinning in a microfuge. A 5 μl aliquot of6-cutter enzyme (EcoRI in this case) was added, and the tube flickedgently to mix, then spun down again. The reaction was incubate at 37° C.for 3 hours, though other times (e.g., about 3-5 hours) could be used.The resultant digest was analyzed by running 2.5 μl on a 1% agarose gel;digestion is illustrated by a DNA smear down.

The digested DNA was extracted using phenol/chloroform, ethanolprecipitated with glycogen, and the resultant pellet resuspended in 20μl low TE (10 mM Tris pH 8.0, 0.1 mM EDTA).

Biotin Linker for EcoR1

Biotin oligos were from Integrated DNA Technologies. The Biotin Linkerspecific for an EcoRI digestion was made from the following twosynthetic oligos (SEQ ID NOs: 1 and 2):

5′-Biotin- TTT CGA CGC TCG CAT CTG GAC AGG-3′ 5′-Phosphate- AAT TCC TGTCCA GAT GCG AGC GTC G-3′The oligonucleotides were allowed to anneal to each other to form BiotinLinker for EcoRI restriction enzyme. On of ordinary skill in the artwill appreciate how to readily vary the sequence of these specificoligonucleotides in order to generate Biotin Linkers for use with otherrestriction enzymes.Ligate Biotin Linkers onto Sticky Ends of Digested Genomic DNA

To the 20 μl sample of restriction digested genomic DNA produced above,5.5 μl (10 μM) of Biotin Linker (specific to the used 6-cutter) wasadded, along with 3 μl 10× T4 ligase buffer w/ ATP. The tube was flickedgently to mix, and the sample spun to the bottom of the tube. Thereaction was incubated at 50° C. for 2 minutes, let sit at roomtemperature (RT; approximate 22° C.) for 10 minutes, and 1.5 μl T4ligase High-Concentration (NEB) added. The tube was flicked gently tomix, the sample spun to the bottom, and incubated at RT for 2-3 hours.The resultant preparation was analyzed by running 2.5 μl on a 1% agarosegel next to diluted biotin linker; free linkers will ligate tothemselves and thereby run slower than the unligated, diluted biotinlinker.

Gel Extract Genomic DNA Away from Free Linkers

Biotinilyated sample prepared as above was run for a short time (˜20minutes at ˜75V) on a 2% TAE low-melt agarose gel, and the highestmolecular weight (top) band comprising the digested genomic DNA withincorporated linkers) was cut out. Care was taken not to harvest the“bottom band” (unincorporated linkers) from the gel. The prepared DNAwas recovered from the low melt agarose using a GELase™ AgaroseGel-Digesting Preparation kit, following the manufacturer's protocol(Epicentre Biotechnologies, Madison, Wis.). The DNA was resuspended in40 μl TE. The resultant preparation was analyzed by running 2.5 μl on a1% agarose gel; correct product is illustrated by a DNA smear down.

Sonication of Linker-Incorporated Digested Genomic DNA

Gel extracted, digested and Tinkered DNA prepared as above was broughtup to a final sample volume of 200 μl with TE, and sonicated using ausing Branson Sonicator 450 (Duty Cycle—80, Output—1.2) as follows: 10pulses, quick spin, 1 minute on ice; repeat 10 times. The resultant,sheared DNA preparation was ethanol precipitated with glycogen, and thepellet resuspended in 40 μl low TE. A 2.5 μl aliquot was run out on a 2%agarose gel to confirm the sonication; the majority of the DNA issheared to ˜200-600 bp when the sonication has been successful.

Pulldown Biotin End-Linked Genomic DNA

The sample was brought to 100 μl with low TE, and reserved while thebeads were prepared. Streptavidin Dynabead solution (50 μl; DynalBiotech, Brown Deer, Wis.) was placed into a separate 1.5 ml tube, towhich was added 150 μl 2× B/W solution (10 mmol/L Tris-HCl, 2 mol/LNaCl, 1 mmol/L EDTA, 1 mL/L Tween 20, pH 7.6). This was pipetted up anddown to mix. A magnet was used to immobilize the beads, and thesupernatant removed. The beads were washed with 200 μl 2× B/W bypipetting up and down, and the wash supernatant removed after the beadswere immobilized with a magnet. The beads were resuspended in 100 μl 2×B/W, and the 100 μl sample added and mixed gently by pipetting up anddown. This was incubated for 15-20 minutes at RT, mixed intermittentlyby pipetting up and down. The beads were immobilized with a magnet, thesupernatant removed, and the wash procedure repeated three times with 1×B/W. The beads were washed once more with 200 μl, then with 200 μl lowTE. The 200 μl TE wash supernatant was saved to be used as a carryovercontrol. After the washing regimen, the beads were resuspended in 85 μllow TE.

Release Genomic DNA Using A 6-Cutter Restriction Endonuclease

To release the prepared tag fragments from the Dynabeads, the DNA wasdigested using the same restriction enzyme used to generate thefragments, as follows. To the 85 μl sample, 10 μl of 10× 6-cutter buffer(in this case, EcoRI) was added and mixed by pipetting up and down. A 5μl aliquot of the 6-cutter enzyme (here, EcoRI) was added and mixed inby pipetting up and down. The mixture was incubated at 37° C. for 1hour, mixing every 10 minutes by pipetting up and down. A magnet wasused to immobilize the now-stripped beads, and the supernatant(containing the prepared DNA tag fragments) removed and placed into anew 1.5 μl tube; the beads were discarded. The DNA preparation wascleaned by phenol/chloroform extraction. By way of example, thefollowing protocol has been used:

Phenol/Chloroform Extraction

-   -   Sample volume was brought to 200 μl w/ TE    -   150 μl Phenol and 150 μl Chloroform were added    -   This was inverted several time, and vortexed well    -   Phases were separated by spinning in a microcentrifuge at max        speed for 2 minutes    -   The top (aqueous) layer was transferred (˜220 μl) to new tube        200 μl Chloroform was added    -   This was inverted several time, and vortexed well    -   Phases were separated by spinning in a microcentrifuge at max        speed for 2 minutes    -   The top (aqueous) layer (˜200 μl) was transferred to new tube

From this point forward, both the sample (“pulldown”) and the low TEwash control were processed. The samples were precipitated with ethanoland glycogen. By way of example, the following protocol has been used:

EtOH Precipitation w/ Glycogen

-   -   The volume of sample to be precipitated was noted    -   1/10 noted volume of 3M NaOAc (pH 7.0) was added    -   The tube was flicked to mix, then the sample brought to the        bottom of the tube with a quick microfuge spin    -   3 μl per 200 μl of sample of 20 μg/μl Glycogen was added    -   The tube was flicked to mix, then the sample brought to the        bottom of the tube 2.5× noted volume of ice cold 100% EtOH        (ethanol) as added    -   The tube was inverted several time to mix, then placed at        −80° C. for 30 minutes DNA was pelleted by spinning at max speed        for 15 minutes at 4° C.    -   The supernatant was removed, the sample spun briefly again, and        any remaining supernatant removed    -   The pellet was washed by adding 1 ml 75% Ice Cold EtOH, vortex        until pellet dislodges from the tube    -   DNA was pelleted by spinning at max speed for 5 minutes at 4° C.    -   The supernatant was removed, the sample spun briefly again, and        any remaining supernatant removed    -   The pellet was allowed to air dry for ˜5 minutes        The resultant pellets resuspended separately in 20 μl TE. 2.5 μl        aliquots of each were run out on a 1% agarose gel for a short        time; DNA was lightly visible in the pulldown sample, but        nothing should be seen in the carryover control.

Random Amplification of Pulldown

The DNA samples were labeled using standard techniques. The following isa representative protocol:

First, the ends of the DNA preparation were repaired. The sample wasbrought to 43 μl in dH₂O in a 1.5 ml tube. To this was added 1 μl (10mM) dNTPs, 5 μl 10× EcoPol buffer, and 1 μl Klenow exo+ (NEB; Beverly,Mass.). The mixture was incubated at 37° C. for 30 minutes, then placedat 75° C. for 10 minutes. The reaction was then purified using a QiagenPCR Purification Kit, eluted in 30 μl EB (provided by the manufacturer).

Blunt-end linkers for use with random amplification were generated byannealing the following oligonucleotides (Integrated DNA Technologies,Coralville, Iowa) (SEQ ID NOs: 3 and 4):

5′- CTG CTC GAA TTC AAG CTT CT-3′ 5′-Phosphate- TCG AGC TGT CCA GAT GCGAGC GTC G-3′The resultant double-stranded blunt-end linkers were added to theresultant fragment preparations as follows: 3.5 μl of dH₂O was placedinto a micro-tube, to which was added 2 μl of end-repaired sample(prepared as above), 2 μl (1 μM) blunt-end linker, 2 μl 10× T4 Ligasebuffer w/ ATP, and 0.5 μl T4 Ligase (High-Concentration, NEB). Thismixture was incubated at Back-RT (˜18° C.) for 2-3 hours.

The blunted ended sample was then amplified using PCR. By way ofexample, the following reaction mixture has been used, in a 1.5 ml tube:

-   -   38.5 μl dH₂O    -   5 μl Thermo Pol buffer    -   1 μl (10 mM) dNTPs    -   0.5 μl NEB Taq    -   2 μl 20 μM Primer (SEQ ID NO: 3)    -   3 μl ligated sample (prepared as above)        Amplification was carried out in a thermocycler, using the        following program cycle        AMPC: 95° C. for 5 minutes; repeat for 20 cycles 90° C. for 45        seconds, 48° C. for 45 seconds, 72° C. for 3 minutes; 72° C. for        5 minutes; 4° C. to stop and store. The resultant amplification        product was purified using a Qiagen PCR Purification Kit, eluted        in 30 μl EB. A 2.5 μl aliquot was run out on a 2% agarose gel;        the majority of the DNA should be ˜200-600 bp. The concentration        of the DNA preparation was now determined using a        spectrophotometer. Preparations usually yielded around 2-4 μg.

Alternatively, amplification could be performed substantially asdescribed in Malrigiorgos et al. (Nature Biotechnology 20:936-939,2002), or other methods that will be known to those of ordinary skill inthe art.

Genomic Labeling and Hybridization

Samples to be run against each other on an array are labeled, forinstance using the following procedures. By way of example, one samplewas labeled with Cy3 and the other with Cy5, though one of ordinaryskill in the art will recognize that other dye pairs (or labelingsystems) could be used.

For each labeling, 0.5 μg of DNA prepared as above was brought to avolume of 10.5 μl w/ dH₂O. To this was added 10 μl 2.5× random primermix (BioPrime Array CGH Purification module, Invitrogen, Carlsbad,Calif.), and the sample was mixed and spun down to the bottom of thetube. The reaction was denatured at 100° C. for 2 minutes, the samplespun back to the bottom of the tube, and placed on ice. To this wasadded:

-   -   2.5 μl 10× dCTP mix    -   1.5 μl Cy3 or Cy5    -   0.5 μl Klenow exo− (Hi-concentration)        The reaction was incubated at 37° c. for 2-5 hours.

The DNA was purified using Qiagen nucleotide removal kit. PN buffer(provided by the manufacturer) was added to each of the two labelingreaction samples, then mixed by pipetting. The two labeling reactionswere then combined and applied to the Bioprime purification column fromInvitrogen (Carlsbad, Calif.). The mixed labeled sample was then elutedin 40 μl EB buffer, and dried under vacuum.

The sample was resuspended in Hyb Mix; 35 μl was used for 22×60 mmlifterslips or 38 μl for 24×60 mm lifterslips, as follows:

Hyb Mixture (Per Slide):

For 22×60 Lifterslips

-   -   20 μl 100% Formamide    -   6 μl 20×SSC    -   4 μl 10% SDS    -   4 μl 50×Denhardt's    -   3.2 μl 10 mg/ml Salmon Sperm DNA

For 24×60 lifterslips

-   -   40 μl 100% Formamide    -   12 μl 20×SSC    -   8 μl 10% SDS    -   8 μl 50×Denhardt's    -   6.4 μl 10 mg/ml Salmon Sperm DNA        The samples (now referred to as probe) were denatured by        incubation at 100° C. for 2 minutes, given a quick spin to        settle the contents to the bottom of the tube, and pipetted onto        the center of the lifterslip after waiting 30 seconds. The        loaded lifterslip was immediately contacted with the slide. The        slide and coverslip were then sealed in Hyb chamber (after        placing 25 μl of 3×SSC in each end), and the hybridization        carried out for 12-16 hours at 42° C.

Wash Protocol

Wash solutions #1, #2, and #3, made fresh for each experiment consistedof the following:

-   -   Wash #1: 380 ml dH₂O, 20 ml 20×SSC, 1.2 ml 10% SDS    -   Wash #2: 198 ml dH₂O, 2 ml 20×SSC    -   Wash #3: 200 ml dH₂O, 0.5 ml 20×SSC        Wash solution #1 was poured into two slide chambers (one wide        and one normal size), using 200 ml in each. Wash solutions #2        and #3 were each poured into a separate slide chamber (normal        size). Slide racks were placed into the normal sized slide        chamber containing wash solution #1 and into the slide chamber        with #2

The Hyb chambers were disassembled and the slide were quickly submerged(keeping the coverslip face up) the wide sized slide chamber thatcontains wash solutions #1 but does not contain a slide rack. Once theslide was submerged, it was tilted to the side and the coverslip felloff immediately. The slide was then quickly transferred into the rack inwash solution #1. The slides were washed in each wash for 2 minutes,plunging up and down several times. Slides were transferred individuallyfrom wash solution #1 into #2; the whole rack (with the slides) wastransferred from #2 to #3. When the slide rack was taken out of wash #3,it was immediately spun for 5 minutes at 600 rpm, then scanned using aGenePix 4000B Microarray Scanner from Axon Instruments (now part ofMolecular Devices).

Results

This example demonstrates that the above protocols can be used toisolate genomic restriction site tags in a non-biased and reproduciblemanner. Genomic restriction site tags were prepared as described abovefrom rucuca and FRT82 flies, using the restriction enzyme EcoRI. Thesetags were labeled with different fluorophores and applied to amicroarray. The two independent trials give the same pattern of yellow(tags present in both genomes), black (tags present in neither genome),red (tags present in the rucuca genome only) and green (tags present inthe FRT82 genome only). This demonstrates that the approach is robustenough to detect the same SNPs in separate trials. The frequency ofdifferently present tags matched our predictions for the Drosophilagenome, indicating that we are not selecting a biased subset of tags.

A feature of SNP detection by this method (and the related approachdescribed, for instance, in Example 2) is that digestion of the genomicDNA with a specific restriction endonuclease creates one set of flankingtags and allows detection of a specific set of SNPs within the sequencescut by that restriction endonuclease. Digestion of the same DNA using adifferent restriction endonuclease will generate a separate set offlanking tags and thereby identify a different group of SNPs. If it isdesired to discover or genotype additional SNPs, then increasing thenumber of digestions with different restriction endonucleases isexpected to increase the number of SNPs found.

We tested this aspect of the system by separately digesting the genomicDNA prepared from the above analysis with XhoI, a 6-cutter restrictionenzyme with a different recognition site sequence than EcoRI. Thispreparation detected a different set of SNPs, as shown by the differentpattern of red, green and yellow spots on the array. Thus, the observeddata closely match the predictions and suggest the isolation of genomictags flanking restriction sites allows for the rapid discovery andgenotyping of SNPs in the genome.

Example 2 Use of Extension from Mismatched Restriction Sites for SNPDiscovery and Mapping

This example provides methods for detecting polymorphism using extensionfrom mismatched restriction sites. A schematic overview of the exemplarymethod is provided in FIG. 6.

Methods

Genomic DNA from two individual flies (referred to as individuals A andB) were separately extracted by using a Blood and cell culture DNA midikit (Qiagen, Valencia, Calif.; cat# 13343). The DNA was completelydigested with the same 4, 5 or 6-cutter restriction enzymes (essentiallyas described above), then purified with a Qiaquick PCR purification kit(Qiagen, Valencia, Calif.; cat# 28106).

10 μg of each of the digested and purified genomic DNA preparations (Aand B) was mixed as a primer and template DNA mixture, and fluorescentlylabeled dCTP or dUTP and unlabeled dNTPs were mixed as a dNTPs resource.A routine PCR reaction was set up. If using Cy5-dCTP to label thereaction, an example of the reaction was set up in a 0.2 ml PCR tube asshown below:

Genomic DNA A: 30 μl Genomic DNA B: 30 μl 10x PCR buffer: 10 μl 10 mMdATP, dTTP, dGTP: 1 μl 4 mM dCTP: 1 μl 1 mM cy5-dCTP: 1 μl Add dH₂O to100 μl 26 μlThis was mixed and 1 units (1 units/μl) DNA taq polymerase added (thoughanother DNA polymerase could be substituted). This was mixed again andthe tube briefly spun in a microcentrifuge to keep all the mixture atthe bottom of the tube. As a negative control, 20 μg genomic A or Balone (annealed to itself) was labeled as above; subtracting signal fromthis negative control allowed removal of noise and signals from, forinstance, transposable elements.

The PCR reaction was carried out in a PCR machine with six cycles of 95°C. one minute and then 72° C. (or 68° C. for some special polymerasesthat prefer 68° C. as an extension temperature, for example HiTaq DNApolymerase from Invitrogen) for 60 minutes. The amplified and labeledreaction mixture was then purified with a Qiaquick PCR purification kit(Qiagen, Valencia, Calif.) and then resuspended in 4×SSC, 1.5 mg/mlpolyA and 0.3% SDS solution.

The preparation was inserted in a boiling water bath for 2 minutes, andthen cooled down for 30 seconds by a brief spinning in amicrocentrifuge. The labeled cDNA mixture was then loaded onto amicroarray slide, covered with a cover slip, put into a hybridizationchamber, and hybridized in a 65° C. water bath for about 16 hours. Afterwashing, the microarray slide was scanned with a Genepix 4000B scanner(Axon Instruments, now Molecular Devices, Union City, Calif.) and thedata was analyzed.

Results

A series of experiments was performed to test the application of thistechnique to SNP discovery. The approach relies on the specificity ofprimer extension at differently cut DNA in the annealed DNA populations.Background extension could result from non-specific shearing of thegenomic DNA, hybridization of related sequences, or incomplete digestionof restriction sites. We first tested if SNPs in a restriction site canbe detected by digestion, annealing, and extension in a simplifiedsystem. High specificity-labeling of DNA was found only at the regionwhere two smaller fragments anneal to a larger fragment (see FIG. 7).

Because the technique worked in the simple setting tested above, a testhybridization using FRT 82 and rucuca genomic DNA annealed after BamHIdigestion was also performed. Labeled material was hybridized to amicroarray made up of overlapping 3 kb clones of Drosophila genomic DNA.One high-intensity spot out of every 30 was observed, similar to whatwould be expected given the rate of polymorphisms in the Drosophilagenome. The rate of background, artifactual extension and hybridizationwas assayed in the context of the full genome by annealing FRT 82genomic DNA cut with BAMHI to itself. We saw strong hybridization toonly a few array elements, indicating that sources of artifactualhybridization are not prevalent in this technique.

This approach has also been used to map a recombination breakpoint.Genomic DNA fragments created and labeled as above from a fly line thatcontains a 3^(rd) chromosome with a mixture of FRT 82 and rucucachromosomal material was annealed to similarly labeled genomic DNAfragments from each of the parental chromosomes in two differentreactions. In chromosomal regions where the recombinant line genomematches the parental line, no extension or subsequent hybridization ispredicted, since the restriction site pattern is the same. Inchromosomal regions where the recombinant DNA comes from the otherparent, the annealed DNA is predicted to produce extension products fromdifferently present restriction sites which would then be detected byhybridization to the array.

This in fact was observed (FIG. 8). In one reaction, the high-intensityspots mapped to the left half of chromosome 3, and only a few spotsmapped to the right side. In the other reaction, high-intensity spotsmapped to the right side. The pattern of hybridization switched modes(from high-intensity spots being present to none, and vice versa) neargenomic position 14,600,000 of the 3^(rd) chromosome. This suggests thatthe recombinant line had a recombination event in this region. Thus, thepresence and absence of restriction sites in the recombinant linecompared to each parental line, as assayed on a microarray, provides arapid method to identify a small chromosomal region of interest.

This disclosure provides various methods for detection of polymorphisms,including particularly the detection of SNPs and other polymorphisms incharacterized and uncharacterized genomes. The disclosure furtherprovides arrays of tags useful in detection of polymorphisms. It will beapparent that the precise details of the methods described may be variedor modified without departing from the spirit of the describedinvention. We claim all such modifications and variations that fallwithin the scope and spirit of the claims below.

1. A method, comprising: isolating fragment tags near restriction sitesfrom genomic DNA by: (a) contacting a preparation of genomic DNA with arestriction endonuclease; (b) subjecting the preparation of genomic DNAto shearing force to fragment the DNA; and (c) isolating DNA fragmentsfrom the resulting preparation based at least in part on their length,which DNA fragments are the fragment tags, wherein (a) and (b) can occurin either order or concurrently; hybridizing the fragment tags from twoindividuals or samples to a collection of nucleic acid elements; anddetecting differential hybridization, where differential hybridizationindicates a sequence difference between the two individuals or samples.2. (canceled)
 3. The method of claim 1, wherein the fragment tags areabout ˜1 kb in length.
 4. The method of claim 1, wherein the collectionof nucleic acid elements is a nucleic acid array or a bead library. 5.The method of claim 1, wherein the collection of nucleic acid elementscomprises genomic DNA fragments, cDNAs or fragments thereof, syntheticnucleic acid sequences, or a combination thereof.
 6. The method of claim1, comprising: shearing genomic DNA to fragments about 3-5 kb in length;filling in any resultant overhanging ends on the fragments; digestingthe sheared DNA with a restriction enzyme, to produce a collection ofDNA fragments at least some of which have a restriction site at one endand a blunt end on the other; isolating from the collection DNAfragments about 1 kb in length; ligating a linker to the sheared DNAfragments about 1 kb in length at the restriction site to producelinkered DNA fragments; isolating Tinkered fragments; and recovering theDNA fragments by digesting away the linker.
 7. A restriction site tagarray or bead library, made by the method of claim
 8. 8. A method ofgenerating a restriction site tag array or bead library, comprising:producing a collection of genomic DNA fragments from an individual, atleast some of which fragments have a restriction site at one end and areblunt at the other by, in either order or concurrently: shearing apreparation of genomic DNA from the individual to fragments about 3-5 kbin length; and digesting the preparation of genomic DNA with arestriction enzyme; isolating DNA fragments about 1 kb in length,thereby producing a tag set; optionally amplifying fragments of the tagset; and using fragments of the tag set as features on the array or inthe bead library.
 9. A subtractive restriction site tag array or beadlibrary, made by the method of claim
 10. 10. A method of making thesubtractive site tag array or bead library, comprising: producing acollection of genomic DNA fragments from each of the two individuals orsamples, at least some fragments of which have a restriction site at oneend and blunt at the other by, in either order or concurrently: shearinga preparation of genomic DNA from each individual to fragments about 3-5kb in length; and digesting each preparation of genomic DNA with arestriction enzyme; isolating DNA fragments about 1 kb in length,thereby producing a tag set from each individual or sample; performingsubtractive hybridization between the two tag sets, to produce acollection of genomic restriction fragment tags that differ between thetwo individuals or samples (differential fragment tags); optionallyamplifying the differential fragment tags; and using the collection ofgenomic restriction fragment tags as features on the array or in thebead library.
 11. A method comprising using the tag array or beadlibrary of claim 7 to detect a nucleotide difference between the genomesof two individuals or between two samples.
 12. A hybridization methodfor polymorphism identification or analysis, comprising hybridizing afirst and a second single-stranded nucleotide molecule, or a first andsecond mixture of single-stranded nucleotide molecules, to the array orbead library of claim 7, and detecting the presence of differentialhybridization.
 13. A hybridization assay comprising: contacting at leastone labeled target nucleic acid sample with an array or bead libraryaccording to claim 7 under conditions sufficient to produce ahybridization pattern; and detecting the hybridization pattern.
 14. Amethod, comprising: annealing to each other restriction enzyme-digestedgenomic DNA fragments from two individuals or samples; adding componentsfor primer extension, in presence of a label capable of being integratedinto resultant extension products; allowing extension along one or moredifferentially cut genomic DNA fragments, which extension incorporatesthe label into the extension products; and detecting labeled extensionproducts, if such are generated, where a labeled extension product isindicative of a sequence difference between the two individuals orsamples.
 15. The method of claim 14, wherein the sequence difference isa nucleotide polymorphism.
 16. A method, comprising: identifying one ormore genomic tags as polymorphic between two individuals or populationsusing the method of claim 1; identifying a tag of interest by selectinga polymorphic spot mapping within the chromosomal region of interest andshowing the presence of a tag in the population not having the trait ofinterest. designing amplification primers, one of which is specific forthe sequence of the tag of interest, the other corresponding to sequencein a DNA adapter sequence ligated to the restriction site; isolatingtags from a bulk population that exhibits the trait of interest; andamplifying the isolated tags using the amplification primers.